Methods of treating lymphoma and leukemia

ABSTRACT

The present invention provides multivalent vaccines for the treatment of B-cell malignancies (e.g., lymphomas and leukemias). The present invention also provides methods for the production of custom vaccines, including multivalent vaccines for the treatment of simmune cell tumors malignancies as well as methods of treating immune cell tumors using custom vaccines.

[0001] This is a Continuation-In-Part of co-pending application Ser. No.08/644,664 filed May 1, 1996.

FIELD OF THE INVENTION

[0002] The present invention generally relates to improved methods forthe amplification and expression of recombinant genes in cells. Theamplified cells provide large quantities of recombinant proteinssuitable for immunotherapy for treatment of lymphomas and leukemias.

BACKGROUND OF THE INVENTION

[0003] As an increasing number of genes are isolated and developed forthe expression of a wide array of useful polypeptide drugs, there is anincreasing need to enhance the efficiencies and economies of theprocess. It is advantageous to obtain such polypeptides from mammaliancells since such polypeptides or proteins are generally correctlyfolded, appropriately modified and completely functional, often inmarked contrast to those proteins as expressed in bacterial cells.

[0004] When large amounts of product are required, it is necessary toidentify cell clones in which the vector sequences are maintained (ie.,retained) during cell proliferation. Such stable vector maintenance canbe achieved either as a consequence of integration of the vector intothe DNA of the host cell or by use of a viral replicon such as bovinepapillomavirus (BPV).

[0005] The use of viral vectors such as BPV-based vectors for thegeneration of stable cell lines expressing large amounts of arecombinant protein has been successful in some cases; however, the useof viral vectors is limited by the fact that the viral vectors arerestricted in the cell types in which they can replicate. Furthermoreexpression levels and episomal maintenance of the viral vector can beinfluenced by the DNA sequences inserted into the vector.

[0006] Where the vector has been integrated into the genomic DNA of thehost cell to improve stability, the copy number of the vector DNA, andconcomitantly the amount of product which could be expressed, can beincreased by selecting for cell lines in which the vector sequences havebeen amplified after integration into the DNA of the host cell.

[0007] A known method for carrying out such a selection procedure is totransform a host cell with a vector comprising a DNA sequence whichencodes an enzyme which is inhibited by a known drug. The vector mayalso comprise a DNA sequence which encodes a desired protein.Alternatively the host cell may be co-transformed with a second vectorwhich comprises the DNA sequence which encodes the desired protein.

[0008] The transformed or co-transformed host cells are then cultured inincreasing concentrations of the known drug hereby selectingdrug-resistant cells. It has been found that one common mechanismleading to the appearance of mutant cells which can survive in theincreased concentrations of the otherwise toxic drug is theover-production of the enzyme which is inhibited by the drug. This mostcommonly results from increased levels of its particular mRNA, which inturn is frequently caused by amplification of vector DNA and hence genecopies.

[0009] It has also been found that when drug resistance is caused by anincrease in copy number of the vector DNA encoding the inhibitableenzyme, there is a concomitant increase in the copy number of the vectorDNA encoding the desired protein in the DNA of the host cell. There isthus an increased level of production of the desired protein.

[0010] The most commonly used system for such co-amplification usesdihydrofolate reductase (DHFR) as the inhibitable enzyme. This enzymecan be inhibited by the drug methotrexate (MTX). To achieveco-amplification, a host cell which lacks an active gene which encodesDHFR is either transformed with a vector which comprises DNA sequencesencoding DHFR and a desired protein or co-transformed with a vectorcomprising a DNA sequence encoding DHFR and a vector comprising a DNAsequence encoding the desired protein. The transformed or co-transformedhost cells are cultured in media containing increasing levels of MTX,and those cell lines which survive are selected.

[0011] The co-amplification systems which are presently available sufferfrom a number of disadvantages. For instance, it is generally necessaryto use a host cell which lacks an active gene encoding the enzyme whichcan be inhibited. This tends to limit the number of cell lines which canbe used with any particular co-amplification system.

[0012] For instance, there are at present, only two cell lines knownwhich lack the gene encoding DHFR and both of these cell lines arederivatives of the CHO-K1 cell line. These DHFR⁻ CHO cell lines cannotbe used to express certain protein products at high levels because CHOcells lack specialized postranslational modification pathways. Forexample, the production of functional human protein C requires that thecell possess the vitamin K-dependent γ-carboxylation pathway; CHO cellscannot properly modify the human protein C protein [Walls et al., (1989)Gene 81:139].

[0013] Attempts to use DHFR genes as dominant selectable markers inother cell lines (i.e., cell lines synthesizing wild type levels ofDHFR) has not proved satisfactory. For instance, a MTX-resistant mutantDHFR or a DHFR gene under the control of a very strong promoter can actas a dominant selectable marker in certain cell types but such highconcentrations of MTX are required that it has not been possible toachieve high copy numbers by selection for gene amplification usingcurrent methodologies.

[0014] Another approach to allow the use of DHFR as a dominantselectable marker in DHFR⁺ cell lines is the use of both the DHFR geneand a gene encoding a selectable marker, such as the hygromycinphosphotransferase (hyg) gene, in addition to the gene of interest[Walls, et al. (1989), supra]. This approach is used to circumvent theproblem of amplification of the endogenous dhfr gene during selectionwith MTX. The cells are transfected with DNA encoding the three genesand the cells are first selected for their ability to grow inhygromycin. The cells are then selected for the ability to grow inincreasing concentrations of MTX. While this approach allows for theco-amplification of genes in dhfr⁺ cell lines, present protocols showthat the dhfr gene is amplified to a higher degree than the gene ofinterest with successive rounds of amplification (i.e., stepwiseincreases in MTX concentration). For example, in several amplifiedclones the dhfr gene was present at approximately 100 copies while thegene of interest was present at only 20 copies.

[0015] Clearly, the art needs improved methods which would consistentlyprovide for the coincidental amplification of the amplifiable marker andthe gene of interest in a variety of cell lines. Furthermore, the artneeds a means of amplifying DNA sequences of interest which isefficient, reproducible and which is not limited to the use ofspecialized enzyme deficient host cell lines or to a limited number ofcell lines.

[0016] Improved methods which consistently provide for the coincidentalamplification of the amplifiable marker and the gene of interest in avariety of cell lines and which are efficient and reproducible wouldallow the production of custom tumor-specific vaccines on a scalecommensurate with patient demand. Current methods for producing customtumor vaccines for the treatment of B-cell lymphoma are insufficient tomeet current and anticipated future demand.

SUMMARY OF THE INVENTION

[0017] The present invention provides methods for the production of celllines containing amplified copies of recombinant DNA sequences. Becausethe amplified cell lines contain several different recombinant DNAsequences (e.g., the amplification vector, one or more expressionvectors and optionally a selection vector) which are coordinatelyamplified, the cell lines are said to have co-amplified the input orexogenous DNA sequences. The methods of the present invention permit theefficient isolation of the desired amplified cell lines with aconsiderable savings in time relative to existing amplificationprotocols. The gene amplification methods of the present inventionpermit the production of custom vaccines, including multivalentvaccines, which are useful for the treatment of immune cell tumors(e.g., lymphomas and leukemias).

[0018] In one embodiment, the present invention provides a multivalentvaccine comprising at least two recombinant variable regions ofimmunoglobulin molecules derived from B-cell lymphoma cells, whereinsaid cells express at least two different immunoglobulin molecules, saidimmunoglobulin molecules differing by at least one idiotope. Theinvention is not limited by the context in which the recombinantvariable regions are utilized; the variable regions may be presentwithin an entire recombinant immunoglobulin (Ig) molecule, they may bepresent on Fab, Fab′ or F(ab′)₂ fragments (which may be generated bycleavage of the recombinant Ig molecule or they may be produced usingmolecular biological means) or they may be present on single chainantibody (Fv) molecules. In a preferred embodiment, the multivalentvaccine comprises at least two recombinant immunoglobulin moleculescomprising said recombinant variable regions derived from said lymphomacells.

[0019] In one embodiment, the immunoglobulin molecules comprisingrecombinant variable regions derived from a patient's lymphoma cells arecovalently linked to an immune-enhancing cytokine. The linkage of thecytokine to the Ig molecule may be achieved by a variety of means knownto the art including conventional coupling techniques (e.g., couplingwith dehydrating agents such as dicyclohexylcarbodiimide (DCCI), ECDIand the like), the use of linkers capable of coupling through sulfhydrylgroups, amino groups or carboxyl groups (available from Pierce ChemicalCo., Rockford, Ill.), by reductive amination. In addition, the covalentlinkage may be achieved by molecular biological means (e.g., theproduction of a fusion protein using an expression vector comprising anucleotide sequence encoding the recombinant Ig operably linked to anucleotide sequence encoding the desired cytokine).

[0020] The invention is not limited by the immune-enhancing cytokineemployed. In a preferred embodiment, the cytokine is selected from thegroup consisting of granulocyte-macrophage colony stimulating factor,interleukin-2 and interleukin-4.

[0021] In one embodiment, the multivalent vaccines of the presentinvention comprise at least one pharmaceutically acceptable excipient.The invention is not limited by the nature of the excipient employed.The pharmaceutical compositions of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiologically bufferedsaline. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0022] In a preferred embodiment, the multivalent vaccine furthercomprises an adjuvant. When the vaccine is to be administered to a humansubject, adjuvants approved for use in humans are employed (e.g., SAF-1,alum, etc.). The recombinant Ig proteins (including fragments of Igproteins) which comprise the multivalent vaccine may be conjugated to acarrier protein such as KLH.

[0023] The present invention also provides a method of producing avaccine for treatment of B-cell lymphoma comprising: a) providing: i)malignant cells isolated from a patient having a B-cell lymphoma; ii) anamplification vector comprising a recombinant oligonucleotide having asequence encoding a first inhibitable enzyme operably linked to aheterologous promoter; iii) a eukaryotic parent cell line; b) isolatingfrom the malignant cells nucleotide sequences encoding at least oneV_(H) region and at least one V_(L) region, the V_(H) and V_(L) regionsderived from immunoglobulin molecules expressed by the malignant cells;c) inserting the nucleotide sequences encoding the V_(H) and V_(L)regions into at least one expression vector; d) introducing theexpression vector(s) and the amplification vector into the parent cellto generate one or more transformed cells; e) growing the transformedcell(s) in a first aqueous solution containing an inhibitor capable ofinhibiting the inhibitable enzyme wherein the concentration of theinhibitor present in the first aqueous solution is sufficient to preventgrowth of the parent cell line; and f) identifying a transformed cellcapable of growth in the first aqueous solution, wherein the transformedcell(s) capable of growth expresses the V_(H) and V_(L) regions. In apreferred embodiment, the transformed cell capable of growth in thefirst aqueous solution contains an amplified number of copies of theexpression vector(s) and an amplified number of copies of theamplification vector.

[0024] In another preferred embodiment, the nucleotide sequencesencoding the V_(H) and C_(L) regions comprise at least two V_(H) and atleast two C_(L) regions (in this manner, a multivalent vaccine isproduced).

[0025] The method of the present invention is not limited by the natureof the means employed to introduce the vectors into the parent cellline. The art is well aware of numerous methods which allow theintroduction of exogenous DNA sequences into mammalian cells, includingbut not limited to electroporation, microinjection, lipofection,protoplast fusion, liposome fusion and the like. In a preferredembodiment, the vectors are introduced into the parent cell line byelectroporation.

[0026] The present invention is not limited by the nature of the cellline chosen as the parent cell line; a variety of mammalian cell linesmay be employed including CHO cell lines and variants thereof, mouse Lcells and BW5147 cells and variants thereof. The chosen cell line growin either an attachment-dependent or attachment-independent manner. In apreferred embodiment, the parent cell line is a T lymphoid cell line; aparticularly preferred T lymphoid cell line is the BW5147.G.1.4 cellline.

[0027] In another embodiment, the method of the present inventionemploys a parent cell line which contains an endogenous gene encoding asecond inhibitable enzyme (e.g., the genome of the parent cell linecontains an endogenous gene comprising a coding region encoding a secondinhibitable enzyme which is operably linked to the promoter naturallylinked to this coding region (ie., the endogenous promoter for thisgene). A contrast is made between the input or exogenous recombinantsequences encoding the first inhibitable enzyme and an endogenous geneencoding an inhibitable enzyme. The endogenous gene sequences will beexpressed under the control of the endogenous promoter. Typically, theamplification vector will comprise a sequence encoding an inhibitableenzyme operably linked to a heterologous (i.e., not the endogenous)promoter. The sequences encoding the first and the second inhibitableenzyme may encode the same or a different enzyme. Furthermore, when thesame enzyme is encoded by the two sequences (i.e., the recombinant andthe endogenous sequences), these sequences may be derived from the sameor a different source (i.e., the recombinant sequence may encode anenzyme isolated from a mouse cell and may introduced into a mouse cellline which contains an endogenous gene encoding the same enzyme;alternatively, the recombinant sequence may encode an enzyme derivedfrom a different species than that of the parent cell line (e.g., therecombinant sequence may encode a rat DHFR and may be introduced into aparent mouse cell line which expresses the mouse DHFR). The amplifiablegene (or marker) and the selectable marker may be present on the samevector; alternatively, they may be present on two separate vectors.

[0028] In one embodiment the second inhibitable enzyme expressed by theparent cell line is selected from the group consisting of dihydrofolatereductase, glutamine synthetase, adenosine deaminase, asparaginesynthetase.

[0029] In another embodiment, the method of the present invention theconcentration of inhibitor present in the first aqueous solution (e.g.,tissue culture medium) used to allow identification of the transformedcell(s) containing amplified copies of the amplification vector andamplified copies of the expression vector(s) is four-fold to six-foldthe concentration required to prevent the growth of the parent cellline. It is well understood by those skilled in the art that only thosesequences present on the amplification vector and expression vector(s)which are required for the expression of the inhibitable enzyme and theprotein(s) of interest, respectively, need to be amplified. However, itis also well understood that any vector backbone sequences linked to thesequences required for expression of the inhibitable enzyme orprotein(s) of interest may also be amplified (and typically are) duringthe co-amplification process.

[0030] In still another embodiment, the method of the present inventionfurther comprises providing a selection vector encoding a selectablegene product (i.e., a selectable marker) which is introduced into saidparent cell line together with said expression vector and saidamplification vector (alternatively, the selectable marker may bepresent on the same vector which contains the amplifiable marker). Theinvention is not limited by the nature of the selectable gene productemployed. The selectable gene product employed may be a dominantselectable marker including but not limited to hygromycin Gphosphotransferase (e.g., the hyg gene product), xanthine-guaninephosphoribosyltransferase (e.g., the gpt gene product) andarninoglycoside 3′ phosphotransferase (e.g., the neo gene product).Alternatively, the selectable marker employed may require the use of aparent cell line which lacks the enzymatic activity encoded by theselectable marker such as hypoxanthine guaninephosphoribosyltransferase, thymidine kinase or carbamoyl-phosphatesynthetase-aspartate transcarbamoylase-dihydrooratase. In a particularlypreferred embodiment, the selection vector encodes an activehypoxanthine guanine phosphoribosyltransferase. When the selectionvector encodes an active hypoxanthine guanine phosphoribosyltransferase,the second aqueous solution which requires the expression of thisselectable gene product comprises hypoxanthine and azaserine.

[0031] In another embodiment, the method of the present inventionfurther comprises following the introduction of the vectors (i.e., theamplification, expression and selection vectors), the additional step ofgrowing the transformed cell in a second aqueous solution which requiresthe expression of the selectable gene product prior to growing thetransformed cell in a first aqueous solution containing an inhibitorcapable of inhibiting said inhibitable enzyme.

[0032] The method of the present invention is not limited by the natureof the inhibitable enzyme encoded by the amplification vector; the artis well of aware of numerous amplifiable markers. In a preferredembodiment, the amplification vector encodes an active enzyme selectedfrom the group consisting of dihydrofolate reductase, glutaminesynthetase, adenosine deaminase, asparagine synthetase.

[0033] In another preferred embodiment, the inhibitor used to select fora transformed cell expressing the inhibitable enzyme encoded by theamplification vector is selected from the group consisting ofmethotrexate, 2′-deoxycoformycin, methionine sulphoximine, albizziin andβ-aspartyl hydroxamate.

[0034] The present invention further provides a method of treatingB-cell lymphoma, comprising: a) providing: i) a subject having a B-celllymphoma; ii) a multivalent vaccine comprising at least two recombinantvariable regions of immunoglobulin molecules derived from the subjects'sB-cell lymphoma cells, wherein the cells express at least two differentimmunoglobulin molecules, the immunoglobulin molecules differing by atleast one idiotope; b) administering said multivalent vaccine to thesubject. In a preferred embodiment, the vaccine comprises at least tworecombinant immunoglobulin molecules comprising the recombinant variableregions derived from the lymphoma cells. In a preferred embodiment, themethod employs a multivalent vaccine which further comprises anadjuvant. When the vaccine is to be administered to a human subject,adjuvants approved for use in humans are employed. In a preferredembodiment the adjuvant is Syntex adjuvant formulation 1. Therecombinant Ig proteins (including fragments of Ig proteins) whichcomprise the multivalent vaccine may be conjugated to a carrier proteinsuch as KLH.

[0035] The present invention provides a method of treating B-celllymphoma, comprising: a) providing: i) a subject having a B-celllymphoma; ii) a multivalent vaccine comprising at least two recombinantvariable regions of immunoglobulin molecules derived from the subjects'sB-cell lymphoma cells, wherein the cells express at least two differentimmunoglobulin molecules, the immunoglobulin molecules differing by atleast one idiotope; and iii) dendritic cells isolated from the subject;b) incubating the dendritic cells in vitro with the multivalent vaccineto produce autologous antigen-pulsed dendritic cells; c) administeringintravenously the pulsed dendritic cells to the subject; and d)following the administration of the pulsed dendritic cells,administering the multivalent vaccine to the subject. In a preferredembodiment, the vaccine comprises at least two recombinantimmunoglobulin molecules comprising the recombinant variable regions.

[0036] The present invention further provides a method of treatingB-cell lymphoma, comprising: a) providing: i) a subject having a B-celllymphoma; ii) a vaccine produced according to the methods of the presentinvention; and b) administering the vaccine to the subject.

[0037] Still further, the present invention provides a method oftreating a subject having an immune cell tumor, comprising: a)providing: i) immune cell tumor cells isolated from a subject, the tumorcells expressing an idiotype protein on the cell membrane; ii) anamplification vector comprising a first recombinant oligonucleotidehaving a sequence encoding a first inhibitable enzyme operably linked toa heterologous promoter; iii) a eukaryotic parent cell line; b)isolating nucleotide sequences encoding at least one idiotype proteinexpressed on the surface of the tumor cells; c) inserting the nucleotidesequences encoding the idiotype protein(s) into at least one vector toproduce at least one expression vector capable of expressing theidiotype protein(s); d) introducing the expression vector(s) into theparent cell to generate one or more transformed cells; e) growing thetransformed cell in a first aqueous solution containing an inhibitorcapable of inhibiting the inhibitable enzyme wherein the concentrationof the inhibitor present in the first aqueous solution is sufficient toprevent growth of the parent cell line; f) identifying a transformedcell capable of growth in the first aqueous solution, wherein thetransformed cell capable of growth contains an amplified number ofcopies of the expression vector and an amplified number of copies of theamplification vector and wherein the transformed cell produces theidiotype protein(s) encoded by the expression vector(s); g) isolatingthe idiotype protein(s) produced by the transformed cell; and h)administering the isolated idiotype protein(s) to the subject.

[0038] The method of the present invention is not limited by the natureof the tumor cells. In one embodiment, the tumor cells are T lymphoidcells and the idiotype protein is a T cell receptor or fragment thereof.In another embodiment, the tumor cells are B lymphoid cells and theidiotype protein is an immunoglobulin or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 shows the map of the expression vector pSSD5. Selectedrestriction enzyme sites are indicated.

[0040]FIG. 2 shows the map of the expression vector pSSD7. Selectedrestriction enzyme sites are indicated.

[0041]FIG. 3 shows the map of the expression vector pSRαSD5. Selectedrestriction enzyme sites are indicated.

[0042]FIG. 4 shows the map of the expression vector pSRαSD7. Selectedrestriction enzyme sites are indicated.

[0043]FIG. 5 shows the map of the expression vector pMSD5. Selectedrestriction enzyme sites are indicated.

[0044]FIG. 6 shows the map of the expression vector pMSD7. Selectedrestriction enzyme sites are indicated.

[0045]FIG. 7 shows the map of the expression vector pHEF1αASD5. Selectedrestriction enzyme sites are indicated.

[0046]FIG. 8 shows the map of the expression vector pHEF1αASD7. Selectedrestriction enzyme sites are indicated.

[0047]FIG. 9 shows the map of the expression vector pHEF1αBSD5. Selectedrestriction enzyme sites are indicated.

[0048]FIG. 10 shows the map of the expression vector pHEF1αBSD7.Selected restriction enzyme sites are indicated.

[0049]FIG. 11 shows the map of the expression vector pMSD5-HPRT.Selected restriction enzyme sites are indicated.

[0050]FIG. 12 shows the map of the expression vector pSSD7-DHFR.Selected restriction enzyme sites are indicated.

[0051]FIG. 13 shows the map of the expression vector pJF14. Selectedrestriction enzyme sites are indicated.

[0052]FIG. 14 shows the map of the expression vector pJFE 14ΔIL10.Selected restriction enzyme sites are indicated.

[0053]FIG. 15 shows the map of the expression vector pSRαSD-DRα-DAF.Selected restriction enzyme sites are indicated.

[0054]FIG. 16 shows the map of the expression vector pSRαSD-DRβ1-DAF.Selected restriction enzyme sites are indicated.

[0055]FIG. 17 is a histogram showing the clone 5 cells selected forgrowth in hypoxanthine and azaserine stained with the L243 monoclonalantibody.

[0056]FIG. 18 is a histogram showing the clone 5 cells selected forgrowth in 80 nM MTX stained with the L243 monoclonal antibody.

[0057]FIG. 19 is a histogram showing the clone 5 cells selected forgrowth in 320 nM MTX stained with the L243 monoclonal antibody.

[0058]FIG. 20 is a histogram showing the clone 5 cells selected forgrowth in 1 μM MTX stained with the L243 monoclonal antibody.

[0059]FIG. 21 shows the map of the expression vector pSRαSD9. Selectedrestriction enzyme sites are indicated.

[0060]FIG. 22 shows the map of the expression vector pSRαSD9CG3C.Selected restriction enzyme sites are indicated.

[0061]FIG. 23 shows the map of the expression vector pSRαSD9CG4C.Selected restriction enzyme sites are indicated.

[0062]FIG. 24 shows the map of the expression vector pSRαSDCKC. Selectedrestriction enzyme sites are indicated.

[0063]FIG. 25 shows the map of the expression vector pSRαSDCL2C.Selected restriction enzyme sites are indicated.

[0064]FIG. 26 shows the map of the selection and amplification vectorpM-HPRT-SSD9-DHFR. Selected restriction enzyme sites are indicated.

DEFINITIONS

[0065] To facilitate understanding of the invention, a number of termsare defmed below.

[0066] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0067] The terms “in operable combination” or “operably linked” as usedherein refers to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the synthesis of adesired protein molecule is produced. When a promoter sequence isoperably linked to sequences encoding a protein, the promoter directsthe expression of mRNA which can be translated to produce a functionalform of the encoded protein. The term also refers to the linkage ofamino acid sequences in such a manner that a functional protein isproduced.

[0068] DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefbre, an end of an oligonucleotides is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region (enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region).Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

[0069] The term “an oligonucleotide having a nucleotide sequenceencoding a gene” means a DNA sequence comprising the coding region of agene or, in other words, the DNA sequence which encodes a gene productThe coding region may be present in either a cDNA or genomic DNA form.Suitable control elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

[0070] The term “recombinant oligonucleotide” refers to anoligonucleotide created using molecular biological manipulations,including but not limited to, the ligation of two or moreoligonucleotide sequences generated by restriction enzyme digestion of apolynucleotide sequence, the synthesis of oligonucleotides (e.g., thesynthesis of primers or oligonucleotides) and the like.

[0071] The term “recombinant oligonucleotide having a sequence encodinga protein operably linked to a heterologous promoter” or grammaticalequivalents indicates that the coding region encoding the protein (e.g.,an enzyme) has been joined to a promoter which is not the promoternaturally associated with the coding region in the genome of an organism(i.e., it is linked to an exogenous promoter). The promoter which isnaturally associated or linked to a coding region in the genome isreferred to as the “endogenous promoter” for that coding region.

[0072] The term “transcription unit” as used herein refers to thesegment of DNA between the sites of initiation and termination oftranscription and the regulatory elements necessary for the efficientinitiation and termination. For example, a segment of DNA comprising anenhancer/promoter, a coding region, and a termination andpolyadenylation sequence comprises a transcription unit.

[0073] The term “regulatory element” as used herein refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defmed infra).

[0074] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes include apromoter, optionally an operator sequence, a ribosome binding site andpossibly other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0075] Transcriptional control signals in eucaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription [Maniatis, et al., Science 236:1237 (1987)].Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect and mammalian cellsand viruses (analogous control elements, i.e., promoters, are also foundin prokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are finctional in a limited subset of cell types [forreview see Voss, et al., Trends Biochem. Sci., 11:287 (1986) andManiatis, et al., supra (1987)]. For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells [Dijkema, et al., EMBO J. 4:761 (1985)]. Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene[Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene91:217 (1990); and Mizushima and Nagata, Nuc. Acids. Res., 18:5322(1990)] and the long terminal repeats of the Rous sarcoma virus [Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)] and the humancytomegalovirus [Boshart et al., Cell 41:521 (1985)].

[0076] The term “promoter/enhancer” denotes a segment of DNA whichcontains sequences capable of providing both promoter and enhancerfunctions (for example, the long terminal repeats of retrovirusescontain both promoter and enhancer functions). The enhancer/promoter maybe “endogenous” or “exogenous” or “heterologous.” An endogenousenhancer/promoter is one which is naturally linked with a given gene inthe genome. An exogenous (heterologous) enhancer/promoter is one whichis placed in juxtaposition to a gene by means of genetic manipulation(i.e., molecular biological techniques).

[0077] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site [Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York (1989) pp. 16.7-16.8]. A commonly used splicedonor and acceptor site is the splice junction from the 16S RNA of SV40.

[0078] Efficient expression of recombinant DNA sequences in eukaryoticcells requires signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation [Sambrook, supra, at 16.6-16.7]. This 237 bpfragment is contained within a 671 bp BamHI/PstI restriction fragment.

[0079] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfcted cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

[0080] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign or exogenous DNA into the genomic DNA ofthe transfected cell.

[0081] The terms “selectable marker” or “selectable gene product” asused herein refer to the use of a gene which encodes an enzymaticactivity that confers resistance to an antibiotic or drug upon the cellin which the selectable marker is expressed. Selectable markers may be“dominant”; a dominant selectable marker encodes an enzymatic activitywhich can be detected in any mammalian cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (also referred to as the neo gene) which confersresistance to the drug G418 in mammalian cells, the bacterial hygromycinG phosphotransferase (hyg) gene which confers resistance to theantibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Other selectablemarkers are not dominant in that their use must be in conjunction with acell line that lacks the relevant enzyme activity. Examples ofnon-dominant selectable markers include the thymidine kinase (tk) genewhich is used in conjunction with TK⁻ cell lines, thecarbamoyl-phosphate synthetase-aspartatetranscarbamoylase-dihydroorotase (CAD) gene which is used in conjunctionwith CAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withHPRT⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook et al., supra at pp.16.9-16.15. It isnoted that some selectable markers can be amplified and therefore can beused as amplifiable markers (e.g., the CAD gene).

[0082] The term “amplification” or “gene amplification” as used hereinrefers to a process by which specific DNA sequences aredisproportionately replicated such that the amplified gene becomespresent in a higher copy number than was initially present in thegenome. Gene amplification occurs naturally during development inparticular genes such as the amplification of ribosomal genes inamphibian oocytes. Gene amplification may be induced by treatingcultured cells with drugs. An example of drug-induced amplification isthe methotrexate-induced amplification of the endogenous dhfr gene inmammalian cells [Schmike et al. (1978) Science 202:1051]. Selection ofcells by growth in the presence of a drug (e.g., an inhibitor of aninhibitable enzyme) may result in the amplification of either theendogenous gene encoding the gene product required for growth in thepresence of the drug or by amplification of exogenous (i.e., input)sequences encoding this gene product, or both.

[0083] The term “co-amplification” as used herein refers to theintroduction into a single cell of an amplifiable marker in conjunctionwith other gene sequences (comprising one or more non-selectable genessuch as those contained within an expression vector) and the applicationof appropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

[0084] The term “amplifiable marker,” “amplifiable gene” or“amplification vector” is used herein to refer to a gene or a vectorencoding a gene which permits the amplification of that gene underappropriate growth conditions. Vectors encoding the dihydrofolatereductase (dhfr) gene can be introduced into appropriate cell lines(typically a dhfr⁻ cell) and grown in the presence of increasingconcentrations of the DHFR inhibitor methotrexate to select for cellswhich have amplified the dhfr gene. The adenosine deaminase (ada) genehas been used in analogous fashion to allow the amplification of adagene sequences in cells selected for growth in the presence of ADAinhibitors such as 2′-deoxycoformycin. Examples of other genes which canbe used as amplifiable markers in mammalian cells include the CAD gene(inhibitor: N-phosphonoacetyl-L-aspartic acid), the ornithinedecarboxylase gene (inhibitor: difluoromethylomithine in medium lackingputrescine), and the asparagine synthetase gene (inhibitors: albizziinor β-aspartyl hydroxamate in asparagine-free medium) [see Kaufman,Methods in Enzymol., 185:537 (1990) for a review].

[0085] The term “gene of interest” as used herein refers to the geneinserted into the polylinker of an expression vector whose expression inthe cell is desired for the purpose of performing further studies on thetransfected cell. The gene of interest may encode any protein whoseexpression is desired in the transfected cell at high levels. The geneof interest is not limited to the examples provided herein; the gene ofinterest may include cell surface proteins, secreted proteins, ionchannels, cytoplasmic proteins, nuclear proteins (e.g., regulatoryproteins), mitochondrial proteins, etc.

[0086] The terms “nucleic acid molecule encoding,” “DNA sequenceencoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0087] The vertebrate hematopoietic system comprises cells of thelymphoid and myeloid lineages. The myeloid lineage (or myeloid-erythroidlineage) gives rise to erythrocytes, basophils, neutrophils,macrophages, eosinophils and platelets. The lymphoid lineage gives riseto B lymphocytes, including plasma cells, and T lymphocytes.

[0088] The term “lymphoid” when used in reference to a cell line or acell, means that the cell line or cell is derived from the lymphoidlineage and includes cells of both the B and the T lymphocyte lineages.

[0089] The terms “T lymphocyte” and “T cell” as used herein encompassany cell within the T lymphocyte lineage from T cell precursors(including Thy1 positive cells which have not rearranged the T cellreceptor genes) to mature T cells (i.e., single positive for either CD4or CD8, surface TCR positive cells).

[0090] The terms “B lymphocyte” and “B cell” encompasses any cell withinthe B cell lineage from B cell precursors, such as pre-B cells (B220⁺cells which have begun to rearrange Ig heavy chain genes), to mature Bcells and plasma cells. “Myeloma” cells or cell lines are malignantplasma cells or cell lines (and are thus in the B cell lineage, not theT cell lineage).

[0091] The terms “parent cell line” or “parental cell line” refers to acell line prior to the addition of exogenous nucleic acid.

[0092] The term “transformed cells” refers to cells which containexogenous DNA (i.e., heterologous DNA introduced into the cells such asthe introduction of an expression vector). Terms “transformed cell” and“transfected cell” are used herein interchangeably.

[0093] The term “aqueous solution” when used in reference to a solutionused to grow a cell line refers to a solution containing compoundsrequired to support the growth of the cells and may contain salts,buffering agents, serum or synthetic serum replacements. An aqueoussolution capable of supporting the growth of a cell line is alsoreferred to as “tissue culture medium” (e.g., EMEM, DMEM, RMPI 1470,etc.).

[0094] An “aqueous solution which requires the expression of aselectable gene product” is a solution or tissue culture medium whichforces a cell line to express a function or active form of theselectable gene product in order for the cells to survive in this medium(e.g., the cell must express a functional HPRT when grown in mediumcontaining hypoxanthine and azaserine). “Aqueous solutions which containan inhibitor capable of inhibiting an inhibitable enzyme” expressed by acell refers to medium containing an inhibitor (e.g., methotrexate) whichis capable of inhibiting an inhibitable enzyme (e.g., DHFR). Thepresence of the inhibitor in the medium requires the cell to express afunctional or active form of the enzyme which is inhibited by theinhibitor in order to survive.

[0095] The “concentration of an inhibitor sufficient to prevent thegrowth of the parent cell line” refers to that concentration ofinhibitor which must be present in the medium to achieve the killing ofgreater than 98% of the cells within 3 to 5 days after plating theparent cells in medium containing the inhibitor.

[0096] The term “amplified number of copies of a vector” refers to acell line which has incorporated an exogenous or recombinant vector andhas increased the number of copies of the vector present in the cell byvirtue of the process of gene amplification.

[0097] The term “amplified gene” refers to a gene present in multiplecopies in a cell line by virtue of gene amplification.

[0098] A cell which contains an “endogenous gene encoding an inhibitableenzyme” refers to cell which naturally (as opposed to by virtue ofrecombinant DNA manipulations) contains in its genomic DNA a geneencoding an inhibitable enzyme; the coding region of this gene will beoperably linked to and under the control of its endogenous promoter.

[0099] The term “active enzyme” refers to an enzyme which is functional(i.e., capable of carrying out the enzymatic finction).

[0100] Immunoglobulin molecules consist of heavy (H) and light (L)chains, which comprise highly specific variable regions at their aminotermini. The variable (V) regions of the H (V_(H)) and L (V_(L)) chainscombine to form the unique antigen recognition or antigen combining siteof the immunoglobulin (Ig) protein. The variable regions of an Igmolecule contain determinants (i.e., molecular shapes) that can berecognized as antigens or idiotypes.

[0101] The term “idiotype” refers to the set of antigenic or epitopicdeterminants (i.e., idiotopes) of an immunoglobulin V domain (i.e., theantigen combining site formed by the association of the complementaritydetermining regions or V_(H) and V_(L) regions).

[0102] The term “idiotope” refers to a single idiotypic epitope locatedalong a portion of the V region of an immunoglobulin molecule.

[0103] The term “anti-idiotypic antibody” or grammatical equivalentsrefers to an antibody directed against a set of idiotopes on the Vregion of an Ig protein.

[0104] A “multivalent vaccine” when used in reference to a vaccinecomprising an idiotypic protein or fragment thereof (e.g.,immunoglobulin molecules or variable regions thereof, T cell receptorproteins or variable regions thereof) refers to a vaccine which containsat least two idiotypic proteins which differ by at least one idiotope.For example, a vaccine which contains two or more immunoglobulinmolecules derived from a B-cell lymphoma where the immunoglobulinmolecules differ from one another by at least one idiotope (e.g., theseimmunoglobulins are somatic variants of one another) is a multivalentvaccine.

[0105] As used herein “recombinant variable regions of immunoglobulinmolecules” refers to variable regions of Ig molecules which are producedby molecular biological means. As shown herein, the variable domain ofthe heavy and light chains may be molecularly cloned from lymphoma cellsand expressed in a host cell (e.g., by insertion into an expressionvector followed by transfer of the expression vector into a host cell);variable domains expressed in this manner are recombinant variableregions of immunoglobulin molecules. The recombinant variable regions ofimmunoglobulin molecules may be expressed as an immunoglobulin moleculecomprising the recombinant variable regions operably linked to theappropriate constant region (i.e., C_(H) or C_(L)) (the constant regionmay comprise the constant region naturally associated with therecombinant variable region, as a Fab, F(ab′)₂ or Fab′ fragmentcomprising the variable domain of the heavy and light chains, theconstant region of the light chain and a portion of the constant regionof the heavy chain (the Fab, F(ab′)₂ or Fab′ fragments may be created bydigestion of a recombinant immunoglobulin molecule or alternatively,they may be produced by molecular biological means), or alternatively,as a single chain antibody or Fv protein.

[0106] “Single-chain antibodies” or “Fv” consist of an antibody lightchain variable domain or region (“V_(L)”) and heavy chain variableregion (“V_(H)”) connected by a short peptide linker. The peptide linkerallows the structure to assume a conformation which is capable ofbinding to antigen [Bird et al., (1988) Science 242:423 and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879].

[0107] A “recombinant variable region derived from a lymphoma cell”refers to a variable region which is molecularly cloned from RNAisolated from a lymphoma cell. The recombinant variable domain may beexpressed as an entire immunoglobulin molecule or may be expressed as afragment of an immunoglobulin molecule, including Fv molecules.

[0108] An “immune-enhancing cytokine” is a cytokine that is capable ofenhancing the immune response when the cytokine is generated in situ oris administered to a mammalian host. Immune-enhancing cytokine include,but are not limited to, granulocyte-macrophage colony stimulating factor(G-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4(IL-4) and interleukin-12 (IL-12).

[0109] An “adjuvant” is a compound which enhances or stimulates theimmune response when administered with an antigen(s).

[0110] “Malignant cells isolated from a patient having a B-celllymphoma” refers to the malignant or pathogenic B-cells found within thesolid tumors characteristic of lymphoma (e.g., lymph nodes and spleencontaining the tumor cells).

DESCRIPTION OF THE INVENTION

[0111] The invention provides vectors and improved methods for theexpression and co-amplification of genes encoding recombinant proteinsin cultured cells. The description is divided into the followingsections: I) Overview of Co-Amplification Methods; II) ExpressionVectors; III) Amplification Vectors; IV) Selection Vectors; V) CellLines and Cell Culture; VI) Co-Transfection of Cell Lines; VII)Selection and Co-Amplification; VIII) Co-Amplification Without PriorSelection; IX) High-Level Expression of Interleukin 10 in Amplified CellLines; X) High-Level Expression of Human Class II MHC Antigens and TCell Receptor Proteins in Amplified Cell Lines; and XI) Production ofCustom Multivalent Vaccines For the Treatment of Lymphoma and Leukemia.

[0112] I. Overview of Co-Amplification Methods

[0113] The present invention provides improved methods for theco-amplification of selectable and non-selectable genes in eukaryoticcell lines. The present invention allows, for the first time, theco-amplification of recombinant gene sequences in T lymphoid cell lines(e.g., the BW5147.G.1.4 cell line).

[0114] The ability to amplify gene sequences in lymphoid cell lines (Tor B lymphoid lines) is desirable for a number of reasons. These includethe ability to of these cells to secrete recombinant immunoglobulins andthe ability to grow these suspension cell lines at high biomass infermentators. To date amplification of input gene sequences has beenreported only in B lymphoid cell lines (e.g., myeloma cell lines).Further, the ability to amplify genes in myeloma cell lines using thedhfr gene as the amplifiable marker have been problematic due to theendogenous DHFR activity in the myeloma cells. Successful amplificationis reported to require the use of a MTX-resistant dhfr gene and the useof very high levels of MTX [Dorai and Moore (1987) J. Immunol.139:4232]. In contrast, the present invention does not require the useof a MTX-resistant dhfr gene and permits the amplification of genes in Tlymphoid cell lines.

[0115] A co-amplification scheme employing the glutamine synthetase (GS)gene has been described [U.S. Pat. No. 5,122,464, the disclosure ofwhich is incorporated by reference herein and Bebbington, et al. (1992)Bio/Technology 10:169]. This co-amplification scheme was developed inpart to circumvent the need to use very high levels of MTX and aMTX-resistant dhfr gene to achieve co-amplification of genes in myelomacells. The use of GS in co-amplification schemes has several drawbacks.First, the propensity of the endogenous GS locus in each cell line to beused must be examined to preclude the use of cell lines in which theendogenous GS locus will amplify at a frequency which makes the GS geneusable. Of four myeloma or hybridoma cell lines, examined, two of thefour (50%) were found to be unsuitable host cells for the use of GS as aselectable marker (Bebbington, et al., supra). One of these twounsuitable cell lines, SP2/0, was found to amplify the endogenous GSlocus.

[0116] A second drawback to the use of GS as a selectable andamplifiable marker is the amount of time required for the isolation ofcell lines producing high levels of the non-selected gene product. Asingle round of amplification and recloning was reported to require 3months using a myeloma cell line subjected to GS selection (Bebbington,et al., supra). Other selectable markers used in co-amplificationprotocols have been reported to require even longer periods of time;selection of amplified myeloma cell lines using DHFR as the selectablemarker takes up to 6 months [Dorai and Moore (1987) J. Immunol.139:4232].

[0117] The present invention provides methods which allow the isolationof the desired amplified cell lines in a shorter period of time thanpermitted using existing co-amplification protocols. Multiple rounds ofamplification can be achieved using the present invention in a period ofabout 3 months. The savings in time is realized, in part, by the use ofcell lines which have rapid doubling times as the host cell line. Inaddition to shortening the period required for the generation of thedesired amplified cell line, the present methods generate with highfrequency amplified cell lines which have co-amplified thenon-selectable gene(s) of interest as well as the amplifiable gene(e.g., the dhfr gene).

[0118] In general the present invention involves the following steps:

[0119] 1. Introduction of linearized plasmids comprising an expressionvector(s) encoding a protein of interest, an amplification vectorencoding an amplifiable marker (e.g., the dhfr gene) and, optionally, aselection vector encoding a selectable marker (e.g., HPRT) into a hostcell line. The host cell line will have a doubling time of 12 hours orless; a particularly preferred host cell line is the BW5147.G.1.4 cellline. The host cell prior to the introduction of the linearized vectorsis referred to as the parental cell line. A preferred means ofintroducing the vector DNA into the host cell line is electroporation.The ratio of the amplification vector, non-selectable expressionvector(s) and/or selection vector is important. A ratio of 1 (selectablevector): 2 (amplification vector): 20-25 [expression vector(s)] isemployed. If a selectable marker is not employed a ratio of 1(amplification vector): 10-15 [expression vector(s)] is used. The use ofthis ratio in conjunction with the electroporation of linearized vectorDNA produces random concatemers of the transfected DNA vectors whichcontain a low percentage of the amplifiable gene. While not limiting theinvention to any particular mechanism, it is believed that these randomconlcatemers containing a low percentage of the amplifiable gene areless likely to generate an amplification unit composed primarily of theamplifiable marker. It is desirable to produce an amplification unitwhich contains primarily the expression vector(s) as this results in anamplified cell line which is expressing large quantities of theprotein(s) of interest.

[0120] In contrast to existing transfection methods (includingelectroporation protocols), the methods of the present invention employlarge quantities of DNA comprising the gene(s) of interest (ie., theexpression vector) [for a discussion of current electroporation methodssee Ausubel et al., Current Protocols in Molecular Biology (1995) JohnWiley & Sons, Inc., at 9.3.1 to 9.3.6]. Using the methods of the presentinvention, a total of about 500 to 750 μg of DNA comprising theexpression vector(s), the amplification vector and if employed, theselection vector in a total volume of 0.5 ml are introduced intoapproximately 2×10⁷ cells in 0.5 of the electroporation buffer (finaldensity of DNA is therefore 1 to 1.5 mg/ml). The use of large quantitiesof the expression vectors increases the frequency with which clones ofcells expressing the gene products encoded by the exogenous DNA areisolated. Using the methods of the present invention about 20 to 25% ofthe selectants (or primary amplificants if no selection vector isemployed) express the genes of interest at relatively high levels. Incontrast, using conventional amounts of DNA (about 20 to 40 μg whenintroducing a single expression vector into the cells), only 1 to 5% ofthe selectants isolated express the gene of interest at relatively highlevels.

[0121] 2. When a selection vector is employed, the transfected cells areallowed to recover by growth in their normal growth medium for a shortperiod (about 36 to 48 hours) and then they are placed in medium whichrequires the cells to express the selectable marker in order to survive(selective medium). The use of the selective medium facilitates theidentification of cells which have taken up the transfected DNA.Colonies of cells which grow in the selective medium (selectants) areexpanded and examined for the ability to express the protein ofinterest. Selectant clones which express the protein(s) of interest athigh levels are then subjected to the amplification process.

[0122] 3. Selectant clones expressing the protein(s) of interest at highlevels are examined to determine their level of sensitivity to theinhibitor which inhibits the enzyme encoded by the amplifiable vector.The sensitivity of the parental cell line to the inhibitor is alsodetermined. Selectants which survive growth in medium containing up to a6-fold higher concentration (typically 4- to 6-fold higher) of theinhibitor than that required to kill the parental cell line are selectedfor fuher manipulation (the first round amplificants). [Any primarytransfectant which has clearly taken up a transfected amplificationvector (e.g., one encoding DHFR) is suitable for continuation with theamplification protocols of the present invention. The presence of thetransfected amplification vector is indicated by the ability of theprimary transfectant to grow in medium containing the inhibitor at alevel which is above the level required to kill the parental cell line.]The first round amplificants are examined for the expression of theprotein(s) of interest. Cells which express low levels of the protein ofinterest are discarded (as this indicates a lack of co-ordinateamplification of the amplifiable gene and the gene(s) of interest).Selectants which are capable of growing in medium containing greaterthan 6-fold the concentration of inhibitor which prevents the growth ofthe parental cell line are discarded. It has been found that selectantswhich are resistant to extremely high levels of the inhibitor typicallydo not yield amplified cell lines which express high quantities of theprotein of interest. While not limiting the present invention to anyparticular mechanism, it is thought that resistance to extremely highlevels of inhibitor at the first round of amplification is indicative ofa cell line in which the amplifiable gene sequences readily separateaway from the majority of the other input DNA sequences (e.g., theexpression vector) resulting the amplification of an amplified unitcomprising primarily the amplifiable gene sequences.

[0123] 4. The first round amplificants which are capable of growing inmedium containing 4-fold to 6-fold higher concentrations of theinhibitor than that required to kill the parental cell line are grown inmedium containing this level of inhibitor for 2 to 3 weeks. The cellsare then grown in medium containing about 4- to 6-fold more of theinhibitor (i.e., 16- to 36-fold the concentration which kills theparental cells) to generate the second round arnplificants. The level ofexpression of the protein(s) of interest are examined in the secondround arnplificants; any clones which do not show an increase inexpression of the protein(s) of interest which corresponds with theincreased resistance to the inhibitor are discarded.

[0124] 5. The amplified cell lines are subjected to subsequent rounds ofamplification by increasing the level of inhibitor in the medium 4- to6-fold for each additional round of amplification. At each round ofamplification, the expression of the protein(s) of interest is examined.Typically any discordance between the level of resistance to theinhibitor and the level of expression of the protein(s) if interest isseen on the second round of amplification. Using the methods of thepresent invention more than 60% of the first round amplificants willco-amplify the gene(s) of interest and the amplifiable gene in thesecond round of amplification. All clones which co-amplified the gene(s)of interest and the amplifiable gene in the second round ofamplification have been found to continue to coordinately amplify thesegene sequences in all subsequent rounds of amplification until a maximumexpression level was reached.

[0125] The following provides additional details regarding the varioussteps and components employed in the co-amplification protocols of thepresent invention.

[0126] II. Expression Vectors

[0127] The expression vectors of the invention comprise a number ofgenetic elements: A) a plasmid backbone; B) regulatory elements whichpermit the efficient expression of genes in eukaryotic cells—theseinclude enhancer/promoter elements, poly A signals and splice junctions;C) polylinkers which allow for the easy insertion of a gene (aselectable marker gene, an amplifiable marker gene or a gene ofinterest) into the expression vector; and D) constructs showing thepossible combination of the genetic elements. These genetic elements maybe present on the expression vector in a number of configurations andcombinations.

A. Plasmid Backbone

[0128] The expression vector contains plasmid sequences which allow forthe propagation and selection of the vector in procaryotic cells; theseplasmid sequences are referred to as the plasmid backbone of the vector.While not intending to limit the invention to a particular plasmid, thefollowing plasmids are preferred. The pUC series of plasmids and theirderivatives which contain a bacterial origin of replication (the pMB1replicon) and the β-lactamase or ampicillin resistance gene. The pUCplasmids, such as pUC18 (ATCC 37253) and pUC19 (ATCC 37254), areparticularly preferred as they are expressed at high copy number(500-700) in bacterial hosts. pBR322 and its derivatives which containthe pMB1 replicon and genes which confer ampicillin and tetracyclineresistance. pBR322 is expressed at 15-20 copies per bacterial cell. pUCand pBR322 plasmids are commercially available from a number of sources(for example, Gibco BRL, Gaithersburg, Md.).

B. Regulatory Elements

[0129] i) Enhancer/Promoters

[0130] The transcription of each cDNA is directed by genetic elementswhich allow for high levels of transcription in the host cell. Each cDNAis under the transcriptional control of a promoter and/or enhancer.Promoters and enhancers are short arrays of DNA which direct thetranscription of a linked gene. While not intending to limit theinvention to the use of any particular promoters and/or enhancerelements, the following are preferred promoter/enhancer elements as theydirect high levels of expression of operably linked genes in a widevariety of cell types. The SV40 and SRα enhancer/promoters areparticularly preferred when the vector is to be transfected into a hostcell which expresses the SV40 T antigen as these enhancer/promotersequences contain the SV40 origin of replication.

[0131] a) The SV40 enhancer/promoter is very active in a wide variety ofcell types from many mammalian species [Dijkema, R. et al., EMBO J.,4:761 (1985)].

[0132] b) The SRa enhancer/promoter comprises the R-U5 sequences fromthe LTR of the human T-cell leukemia virus-1 (HTLV-1) and sequences fromthe SV40 enhancer/promoter [Takebe, Y. et al., Mol. Cell. Biol., 8:466(1988)]. The HTLV-1 sequences are placed immediately downstream of theSV40 early promoter. These HTLV-1 sequences are located downstream ofthe transcriptional start site and are present as 5′ nontranslatedregions on the RNA transcript. The addition of the HTLV-1 sequencesincreases expression from the SV40 enhancer/promoter.

[0133] c) The human cytomegalovirus (CMV) major immediate early gene(IE) enhancer/promoter is active in a broad range of cell types [Boshartet al., Cell 41:521 (1985)]. The 293 cell line (ATCC CRL 1573) [J. Gen.Virol., 36:59 (1977), Virology 77:319 (1977) and Virology 86:10 (1978)],an adenovirus transformed human embryonic kidney cell line, isparticularly advantageous as a host cell line for vectors containing theCMV enhancer/promoter as the adenovirus IE gene products increase thelevel of transcription from the CMV enhancer/promoter.

[0134] d) The enhancer/promoter from the LTR of the Moloney leukemiavirus is a strong promoter and is active in a broad range of cell types[Laimins et al., Proc. Natl. Acad. Sci. USA 79:6453 (1984)].

[0135] e) The enhancer/promoter from the human elongation factor 1α geneis abundantly transcribed in a very broad range of cell types [Uetsukiet al., J. Biol. Chem., 264:5791 (1989) and Mizushima and Nagata, Nuc.Acids. Res. 18:5322 (1990)].

[0136] ii) Poly A Elements

[0137] The cDNA coding region is followed by a polyadenylation (poly A)element. The preferred poly A elements of the present invention arestrong signals that result in efficient termination of transcription andpolyadenylation of the RNA transcript. A preferred heterologous poly Aelement is the SV40 poly A signal (See SEQ ID NO:3). Another preferredheterologous poly A element is the poly A signal from the humanelongation factor 1α (hEF1α) gene. (See SEQ ID NO:41). The invention isnot limited by the poly A element utilized. The inserted cDNA mayutilize its own endogenous poly A element provided that the endogenouselement is capable of efficient termination and polyadenylation.

[0138] iii) Splice Junctions

[0139] The expression vectors also contain a splice junction sequence.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site. The presenceof splicing signals on an expression vector often results in higherlevels of expression of the recombinant transcript. A preferred splicejunction is the splice junction from the 16S RNA of SV40. Anotherpreferred splice junction is the splice junction from the hEF1α gene.The invention is not limited by the use of a particular splice junction.The splice donor and acceptor site from any intron-containing gene maybe utilized.

C. Polylinkers

[0140] The expression vectors contain a polylinker which allows for theeasy insertion of DNA segments into the vector. A polylinker is a shortsynthetic DNA fragment which contains the recognition site for numerousrestriction endonucleases. Any desired set of restriction sites may beutilized in a polylinker. Two preferred polylinker sequences are the SD5and SD7 polylinker sequences. The SD5 polylinker is formed by the SD5A(SEQ ID NO:1) and SD5B (SEQ ID NO:2) oligonucleotides and contains therecognition sites for XbaI, NotI, SfiI, SacII and EcoRI. The SD7polylinker is formed by the SD7A (SEQ ID NO:4) and SD7B (SEQ ID NO:5)oligonucleotides and contains the following restriction sites: XbaI,EcoRI, MluI, StuI, SacII, SfiI, NotI, BssHII and SphI. The polylinkersequence is located downstrearn of the enhancer/promoter and splicejunction sequences and upstream of the poly A sequence. Insertion of acDNA or other coding region (i.e., a gene of interest) into thepolylinker allows for the transcription of the inserted coding regionfrom the enhancer/promoter and the polyadenylation of the resulting RNAtranscript.

D. Constructs

[0141] The above elements may be arranged in numerous combinations andconfigurations to create the expression vectors of the invention. Thegenetic elements are manipulated using standard techniques of molecularbiology known to those skilled in the art [Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York (1989)]. Once a suitable recombinant DNAvector has been constructed, the vector is introduced into the desiredhost cell. DNA molecules are transfected into procaryotic hosts usingstandard protocols. Briefly the host cells are made competent bytreatment with calcium chloride solutions (competent bacteria cells arecommercially available and are easily made in the laboratory). Thistreatment permits the uptake of DNA by the bacterial cell. Another meansof introducing DNA into bacterial cells is electroporation in which anelectrical pulse is used to permit the uptake of DNA by bacterial cells.

[0142] Following the introduction of DNA into a host cell, selectivepressure may be applied to isolate those cells which have taken up theDNA. Procaryotic vectors (plasmids) will contain anantibiotic-resistance gene, such as ampicillin, kanamycin ortetracycline resistance genes. The preferred pUC plasmids contain theampicillin resistance gene. Growth in the presence of the appropriateantibiotic indicates the presence of the vector DNA.

[0143] For analysis to confirm correct sequences in the plasmidsconstructed, the ligation mixture may be used to transform suitablestrains of E. coli. Examples of commonly used E. coli strains includethe HB101 strain (Gibco BRL), TG1 and TG2 (derivatives of the JM101strain), DH10B strain (Gibco BRL) or K12 strain 294 (ATCC No. 31446).Plasmids from the transformants are prepared, analyzed by digestion withrestriction endonucleases and/or sequenced by the method of Messing etal., Nuc. Acids Res., 9:309 (1981).

[0144] Plasmid DNA is purified from bacterial lysates by chromatographyon Qiagen Plasmid Kit columns (Qiagen, Chatsworth, Calif.) according tothe manufacturer's directions for large scale preparation.

[0145] Small scale preparation (i.e., minipreps) of plasmid DNA isperformed by alkaline lysis [Birnboim, H. C. and Doly, J., Nuc. Acids.Res., 7:1513 (1979)]. Briefly, bacteria harboring a plasmid is grown inthe presence of the appropriate antibiotic (for pUC-based plasmidsampicillin is used at 60 μg/ml) overnight at 37° C. with shaking. 1.5 mlof the overnight culture is transferred to a 1.5 ml microcentrifugetube. The bacteria are pelleted by centrifugation at 12,000 g for 30seconds in a microcentrifuge. The supernatant is removed by aspiration.The bacterial pellet is resuspended in 100 μl of ice-cold Solution I (50mM glucose, 25 mM Tris-HCl, pH 8.0 and 10 mM EDTA, pH 8.0). Two hundredμl of Solution II (0.2 N NaOH and 1% SDS) is added and the tube isinverted to mix the contents. 150 μl of ice-cold Solution III (3M sodiumacetate adjusted to pH 4.8 with glacial acetic acid) is added and thetube is vortexed to mix the contents. The tube is then placed on ice for3 to 5 minutes. The tube is then centrifuged at 12,000g for 5 minutes ina microcentrifuge and the supernatant is transferred to a fresh tube.The plasmid DNA is precipitated using 2 volumes of ethanol at roomtemperature and incubating 2 minutes at room temperature (approximately25° C.). The DNA is pelleted by centrifugation at 12,000 g for 5 minutesin a microcentrifuge. The supernatant is removed by aspiration and theDNA pellet is resuspended in a suitable buffer such as TE buffer (10 mMTris-HCl, pH 7.6, 1 mM EDTA, pH 8.0).

[0146] Expression vector DNA purified by either chromatography on Qiagencolumns or by the alkaline lysis miniprep method is suitable for use intransfection experiments.

[0147] III. Amplification Vectors

[0148] A vector encoding a structural gene which permits the selectionof cells containing multiple or “amplified” copies of the vectorencoding the structural gene is referred to as an amplification vector.The amplifiable gene is capable of responding either to an inhibitor orlack of an essential metabolite by amplification to increase theexpression product (i.e., the expression of the protein encoded by theamplifiable gene). The amplifiable gene may be characterized as beingable to complement an auxotrophic host. For example, the gene encodingDHFR may be used as the amplifiable marker in conjunction with cellslacking the ability to express a functional DHFR enzyme. However, it isnot necessary to use an auxotrophic host cell. In a preferred embodimentthe host cell is not auxotrophic with respect to the amplifiable marker.

[0149] The invention is not limited by the use of a particularamplifiable gene. Various genes may be employed, such as the geneexpressing DHFR, the CAD gene, genes expressing metallothioneins, thegene expressing asparagine synthetase, the gene expressing glutaminesynthetase and genes expressing surface membrane proteins which offerdrug resistance. By blocking a metabolic process in the cells withenzyme inhibitors, such as methotrexate, for DHFR or cytotoxic agentssuch as metals, with the metallothionein genes, or by maintaining a lowor zero concentration of an essential metabolite, the cellular responsewill be amplification of the particular gene and flanking sequences[Kaufman and Sharp (1982) J. Mol. Biol. 159:601]. Because the process ofgene amplification results in the amplification of the amplifiablemarker and surrounding DNA sequences, it is possible to co-amplify genesequences other than those encoding the amplifiable marker [Latt, et al.(1985) Mol. Cell. Biol. 5:1750]. The amplification of sequences encodingthe gene of interest is accomplished by co-introducing sequencesencoding the gene of interest and the amplifiable marker into the samehost cell.

[0150] The gene encoding the protein of interest may be physicallylinked to the amplifiable marker by placing both coding regions withappropriate regulatory signals on a single vector. However it is notnecessary that both coding regions be physically located on the samevector. Because small vector molecules are easier to manipulate and givehigher yields when grown in bacterial hosts, it is preferred that thegene of interest and the amplifiable marker gene be located on twoseparate plasmid vectors. Whether the amplifiable marker and the gene ofinterest are encoded on the same or separate vector plasmids, the vectormolecules are linearized by digestion with a restriction enzyme prior tointroduction of the vector DNAs into the host cell. The restrictionenzyme utilized is selected for its ability to cut within the plasmidbackbone of the vector but not cut within the regulatory signals or thecoding region of the amplifiable marker or gene of interest.

[0151] The amplification vector is constructed by placing the desiredstructural gene encoding the amplifiable marker into an expressionvector such that the regulatory elements present on the expressionvector direct the expression of the product of the amplifiable gene. Theinvention is illustrated by the use of a structural gene encoding DHFRas the amplifiable marker. The DHFR coding sequences are placed in thepolylinker region of the expression vector pSSD7 such that the DHFRcoding region is under the transcriptional control of the SV40enhancer/promoter. The invention is not limited by the selection of anyparticular vector for the construction of the amplification vector. Anysuitable expression vector may be utilized. Particularly preferredexpression vectors include pSSD5, pSSD7, pSRαSD5, pSRαSD7, pMSD5 andpMSD7. These expression vectors utilize regulatory signals which permithigh level expression of inserted genes in a wide variety of cell types.

[0152] IV. Selection Vectors

[0153] An expression vector encoding a selectable marker gene isreferred to as a selection vector. The selectable marker may be adominant selectable marker. Examples of dominant selectable markersinclude the neo gene, the hyg gene and the gpt gene. The selectablemarker may require the use of a host cell which lacks the ability toexpress the product encoded by the selectable marker. Examples of suchnon-dominant markers include the tk gene, the CAD gene and the hprtgene.

[0154] The invention is not limited to the use of a particularselectable marker or to the use of any selectable marker (besides theamplifiable marker) at all. In a preferred embodiment, the host cellused is a HPRT-deficient cell line and the amplifiable marker used isDHFR.

[0155] When an HPRT-deficient cell line is utilized and this cell lineproduces a functional DHFR enzyme, a selectable marker encoding the HPRTenzyme may be utilized. The host cell is co-transfected with plasmidscontaining a selectable marker (HPRT), an amplifiable marker (DHFR) andone or more proteins of interest. The transfected cells are then firstselected for the ability to grow in HxAz medium (hypoxanthine andazaserine) which requires the expression of HPRT by the cell. Cellswhich have the ability to grow in HxAz medium have incorporated at leastthe selection vector encoding HPRT. Because the vector DNAs arelinearized and then introduced into the host cell by electroporation(discussed below), cells which have taken up the HPRT vector are alsolikely to have taken up the vectors encoding DHFR and the protein ofinterest. This is because the linearized vectors form long concatemersor tandem arrays which integrate with a very high frequency into thehost chromosomal DNA as a single unit [Toneguzzo, et al. (1988) Nucl.Acid Res. 16:5515].

[0156] The ability to select for transfected cells expressing HPRTfacilitates the use of DHFR as the amplifiable marker in a cell linewhich is not DHFR-deficient. The use of the selectable marker allows thecircumvention of the problem of amplification of the host cell'sendogenous DHFR gene [Walls, J. D. et al., (1989), supra]. However, asdiscussed below, the present invention can be practiced without using aselectable marker in addition to the amplification vector when celllines which are not DHFR-deficient are employed.

[0157] The invention may be practiced such that no selectable marker isused. When the amplifiable marker is a dominant amplifiable marker suchas the glutamine synthetase gene or where the host cell line lacks theability to express the amplifiable marker (such as a DHFR⁻ cell line) noselectable marker need be employed.

[0158] V. Cell Lines And Cell Culture

[0159] A variety of mammalian cell lines may be employed for theexpression of recombinant proteins according to the methods of theresent invention. Exemplary cell lines include CHO cell lines [e.g.,CHO-K1 cells (ATCC CCl 61; ATCC CRL 9618) and derivations thereof suchas DHFR⁻ CHO-KI cell lines (e.g., CHO/dhFr-; ATCC CRL 9096), mouse Lcells and BW5147 cells and variants thereof such as BW5147.3 (ATCC TIB47) and BW5147.G.1.4 cells (ATCC TIB 48). The cell line employed maygrow attached to a tissue culture vessel (i.e, attachment-dependent) ormay grow in suspension (i.e., attachment-independent).

[0160] BW5147.G.1.4 cells are particularly preferred for the practice ofthe present invention. BW5147.G.1.4 cells have a very rapid doublingtime [i.e., a doubling time of about 12 hours when grown in RPMI 1640medium containing 10% Fetal Clone I (Hyclone)]. The doubling time orgeneration time refers to the amount of time required for a cell line toincrease the number of cells present in the culture by a factor of two.In contrast, the CHO-K1 cell line (from which the presently availabledhfr- CHO-KI cell lines were derived) have a doubling time of about 21hours when the cells were grown in either DMEM containing 10% FetalClone II (Hyclone) or Ham's F-12 medium containing 10% Fetal Clone II.

[0161] A rapid doubling time is advantageous as the more rapidly a cellline doubles, the more rapidly amplified variants of the cell line willappear and produce colonies when grown in medium which requires theexpression of the amplifiable marker. Small differences (i.e., 1-2hours) in the doubling times between cell lines can translate into largedifference in the amount of time required to select for a cell linehaving useful levels of amplification which result in a high level ofexpression of the non-selectable gene product. The speed with which ahigh expressing cell line can be isolated may be critical in certainsituations. For example, the production of proteins to be used inclinical applications (e.g., the production of tumor-related proteins tobe used to immunize a cancer patient) requires that the protein ofinterest be expressed in useable quantities as quickly as possible sothat maximum benefit to the patient is realized.

[0162] In addition, BW5147.G.1.4 cells permit the amplification of thenon-selectable gene (which encodes the protein of interest) at a veryhigh fiequency. Using the methods of the present invention, about 80% ofBW5147.G. 1.4 cells which survive growth in the selective medium (e.g.,HxAz medium) will amplify the input DNA which contains the amplifiablemarker and the DNA encoding the protein of interest (as measure by theability of the cells to survive in medium containing MTX and theproduction of increased amounts of the protein of interest). That is 80%of the cells which survive growth in the selective medium will survivegrowth in medium which requires the expression of the amplifiablemarker. When cells are subjected to growth in medium containing acompound(s) which requires expression of the amplifiable marker (e.g.,growth in the presence of MTX requires the expression of DHFR), thecells which survive are said to have been subjected to a round ofamplification. Following the initial or first round of amplification,the cells are placed in medium containing an increased concentration ofthe compounds which require expression of the amplifiable marker and thecells which survive growth in this increased concentration are said tohave survived a second round of amplification. Another round ofselection in medium containing yet a further increase in theconcentration of the compounds which require expression of theamplifiable marker is referred to as the third round of amplification.

[0163] Of those transfected BW5147.G.1.4 clones which amplify in thefirst round of amplification (as measured by both the ability to grow inincreased concentrations of MTX and an increased production of theprotein of interest), about ⅔ also coordinately amplify the amplifiablegene as well as the gene encoding the protein of interest in the secondround of amplification. All clones which coordinately amplified theamplifiable marker and the gene encoding the protein of interest in thesecond round of amplification have been found to coordinately amplifyboth genes in all subsequent rounds of amplification.

[0164] An additional advantage of using BW5147.G.1.4 cells is the factthat these cells are very hardy. A cell line is said to be hardy when itis found to be able to grow well under a variety of culture conditionsand when it can withstand a certain amount of mal-treatment (i.e., theability to be revived after being allowed to remain in medium which hasexhausted the buffering capacity or which has exhausted certainnutrients). Hardiness denotes that the cell line is easy to work withand it grows robustly. Those skilled in the art of tissue culture knowreadily that certain cell lines are more hardy than others; BW5147.G.1.4cells are particularly hardy cells.

[0165] BW5147.G.1.4 cells may be maintained by growth in DMEM containing10% FBS or RPMI 1640 medium containing 10% Fetal Clone I. CHO-K1 cells(ATCC CCl 61, ATCC CRL 9618) may be maintained in DMEM containing 10%Fetal Clone II (Hyclone), Ham's F12 medium containing 10% Fetal Clone IIor Ham's F12 medium containing 10% FBS and CHO/dhFr- cells (CRL 9096)may be maintained in Iscove's modified Dulbecco's medium containing 0.1mM hypoxanthine, 0.01 mM thymidine and 10% FBS. These cell lines aregrown in a humidified atmosphere containing 5% CO₂ at a temperature of37° C.

[0166] The invention is not limited by the choice of a particular hostcell line. Any cell line can be employed in the methods of the presentinvention. Cell lines which have a rapid rate of growth or a lowdoubling time (ie., a doubling time of 15 hours or less) and which iscapable of amplifying the amplifiable marker at a reasonable ratewithout amplification of the endogenous locus at a similar or higherrate are preferred. Cell lines which have the ability to amplify theamplifiable marker at a rate which is greater than the rate at which theendogenous locus is amplified are identified by finding that the abilityof the cell to grow in increasing concentrations of the inhibitor (i.e.,the compound which requires the cell to express the amplifiable markerin order to survive) correlates with an increase in the copy number ofthe amplifiable marker (this may be measured directly by demonstratingan increase in the copy number of the amplifiable marker by Southernblotting or indirectly by demonstrating an increase in the amount ofmRNA produced from the amplifiable marker by Northern blotting).

[0167] VI. Co-Transfection Of Cell Lines

[0168] Prior to introduction of vector DNA into a given cell line, thevector DNA is linearized using a restriction enzyme which cuts oncewithin the vector sequences and which does not cut within the control orcoding regions necessary for the expression of the encoded protein.Linearization of the DNA is advantageous as it promotes the integrationof the vector DNA into the chromosomal DNA of the host cell line (freeends of DNA are recombinogenic). Furthermore, vector DNA must break inorder to integrate into the genomic DNA of the host cell; linearizationallows control over where this break occurs thereby preventing the lossof finctional vector sequences by directing this break to anon-essential region of the vector. Additionally, linear DNA moleculestend to integrate into the genomic DNA of the host cell as a random headto tail concatemer (it is noted that circular DNA also tends tointegrate as a head to tail concatemer; however, as discussed above, thecircular DNA must break prior to integration). This obviates the need toconstruct a single large vector containing the selectable gene,amplifiable gene and the gene(s) of interest. Several smaller vectorsmay be co-transfected instead thereby essentially eliminating thelikelihood that the vector will suffer a break in an essential region.

[0169] To generate a stable cell line expressing large quantities of adesired protein(s), the following vectors are introduced as linearDNA: 1) a selectable vector such as pMSD5-HPRT; 2) an amplifiable vectorsuch as pSSD7-DHFR and 3) one or more vectors encoding a gene ofinterest. This also results in a much higher ration of copies of theexpressed gene(s) of interest to amplifiable marker genes in theconcatemer. The ratio of the selectable vector, amplifiable vector andthe vector(s) encoding a protein(s) of interest is 1:2:20-50. Multiplevectors encoding separate proteins of interest are utilized when it isdesirable to express multiple proteins in a single cell. This will bethe case where the protein of interest is a multi-chain protein. Forexample, immunoglobulins are formed by the association of two heavychains and two light chains; the heavy and light chains are encoded byseparate genes. Expression of a functional immunoglobulin requires thatthe transfected cell express both the heavy and light chain genes. Up tosix non-selectable/amplifiable plasmids (i.e., encoding a gene ofinterest) may be used to transfect a given cell line.

[0170] Large quantities of the expression vector(s) are introduced intothe cells along with the amplification and selection vectors. Typically10 to 15 μg of the selectable vector (e.g., pMSD5-HPRT), 20 to 30 μg ofthe amplification vector (e.g., pSSD7-DHFR) and a total of 400 to 500 μgtotal of the expression vectors. For example, when two expressionvectors are to be used, 200 to 250 μg of each of the two expressionvectors (ie., plasmid encoding a gene of interest) are used in additionto the selection and amplification vectors. The maximum amount of DNAwhich can be electroporated under the conditions used herein is about500 to 750 μg DNA (i.e., the total amount or the sum of all vectorDNAs). If 6 separate expression vectors are to be introduced into a cellin addition to the selection and amplification vectors, the followingamounts of DNA are employed: 7.5 μg of the selection vector, 15 μg ofthe amplification vector and ˜121 μg of each of the six expressionvectors [the total amount of DNA is therefore ˜750 μg perelectroporation using 2×10⁷ cells/ml in 0.5 ml of 1X HBS(EP)].

[0171] The vectors to be co-transfected into the cells are linearizedusing appropriate restriction enzymes (i.e., enzymes which cut onlywithin the plasmid backbone) in the same reaction tube. Followingdigestion with the appropriate restriction enzymes, the DNA isprecipitated using ethanol and resuspended in 0.5 ml of 1X HBS (EP) (20mM HEPES, pH 7.0; 0.75 mM Na₂HPO₄/NaH₂PO₄, pH 7.0; 137 mM NaCl; 5 mM KCland 1 gm/liter dextrose).

[0172] The linearized vector DNAs are preferentially introduced into thehost cell by electroporation. Alternatively, the linearized vector DNAsmay be introduced into the host cell by microinjection using techniquesknown to the art. The use of electroporation is preferred over othermethods of introducing DNA into cells for a number of reasons: 1)efficiency of transfection. A number of attractive cell lines (e.g.,virtually any lymphoid cell line) are refractory to transformation viaany other method (such as DEAE-dextran mediated transfection or calciumphosphate-DNA co-precipitation). Electroporation of these lines allowsthe ready isolation of as many independent transformants as might bereasonably required. 2) Electroporation preserves the integrity of thetransfected DNA. DNA introduced by other methods (DEAE-dextran or CaPO₄)has been shown to acquire observable mutations at observablefrequencies, posing a concern for therapeutically used proteins derivedfrom these sorts of transfections [See for example, M. P. Calos et al.(1983) Proc. Natl. Acad. Sci. USA 80:3015; Kopchick and Stacey (1984)Mol. Cell. Biol. 4:240; Wake et al. (1984) Mol. Cell. Biol. 4:387; andLebkowski et al. (1984) Mol. Cell. Biol. 4:1951]. Lebkowski et al.,supra reported a mutation frequency in DNA chemically introduced thatwas four orders of magnitude above the endogenous mutational frequency.In contrast, DNA introduced into cells via electroporation was found tohave a mutation frequency equal to the background mutational frequencyof the cell [Drinkwater and Klinedinst (1986) Proc. Natl. Acad. Sci. USA83:3402].

[0173] 3) Cotransformation of several unlinked DNA molecules is readilyachieved using electroporation. As demonstrated herein, a minimum offour unlinked DNAs can be cotransfected into cells by electroporationand a high frequency of the cells expressing the selectable marker willalso express all of the other genes. 4) Electroporation is simple toperform. While microinjection of DNA avoids the increased mutationfrequency observed using chemical introduction of DNA, microinjection ofsomatic cells is technically challenging and requires the use ofexpensive equipment. In contrast electroporation can be performed usingfairly inexpensive equipment which may be prepared in house or purchasedcommercially.

[0174] Lymphoid cell lines have been very difficult to transfect withCaPO₄-mediated co-precipitation, although it has been achieved [Rice andBaltimore (1982) Proc. Natl. Acad. Sci. USA 79:7862 and Oi et al. (1983)Proc. Natl. Acad. Sci. USA 80:825]. In contrast, transfection ofnumerous lymphoid cell lines has been achieved by electroporation withacceptably high transformation frequencies [Potter et al. (1984) Proc.Natl. Acad. Sci. USA 81: 7161; Boggs et al. (1986) Exp. Hematol. 14:988;Toneguzzo et al. (1986) Mol. Cell. Biol. 6:703 and Toneguzzo and Keating(1986) Proc. Natl. Acad. Sci. USA 83:3496]. Oi et al., supra report atransformation frequency for BW5147 cells using CaPO₄-mediatedco-precipitation and a gpt-expressing plasmid of 1 per 10⁷ cells.Toneguzzo et al., supra report a transformation frequency for BW5147cells using electroporation and a gpt-expressing plasmid of 3.6 per 10⁴cells (a frequency greater than 3000-fold higher than that achievedusing CaPO₄-mediated co-precipitation).

[0175] The host cells, typically BW5147.G.1.4 cells, are washed twice inice-cold 1X HBS(EP) and resuspended at 2×10⁷ cells/ml in 0.5 ml of 1X.HBS(EP). The cells are then placed in a 1 ml cuvette (#67.746, Sarstedt,Inc., Princeton, N.J.) which contains the linearized DNAs. The cuvetteis placed on ice. The electroporation is performed at 225 volts using anISCO Model 493 power supply (ISCO). The electroporation apparatus isconstructed exactly as described in Chu, G. et al., Nucl. Acids Res.15:1311 (1987). The electroporation device is set on constant voltage(225V) at the 2X setting (i.e., both capacitors are used).Alternatively, a commercially available electroporation device may beemployed [e.g., Gene Pulser™ (BioRad, Hercules, Calif.) with theCapacitance Extender set at 960 μFD]. Following electroporation, thecells are allowed to recover by incubation on ice for 5 to 15 minutes,typically 10 minutes.

[0176] VII. Selection And Co-Amplification

[0177] The electroporated cells are then transferred to a T75 flask(Falcon) containing 30 mls of RPMI 1640 medium (Irvine Scientific)supplemented with 10% fetal calf serum (FCS; HyClone) and 50 μg/mlgentamicin (Sigma). The cells are then incubated at 37° C. in ahumidified atmosphere containing 5% CO₂ for 36 to 48 hours. The cellsare then transferred to 48 well plates (Costar) at 1×10⁴ to 1×10⁵ cellsper well in selective medium. The use of selective medium facilitatesthe identification of cells which have taken up the transfected DNA.Cells which grow either in an attachment-dependent manner or anattachment-independent manner are plated in multiwell plates duringgrowth in selective medium.

[0178] A variety of selectable markers may be used including bothdominant selectable markers and markers which require the use of a cellline lacking a given enzyme. For example, cell lines lacking the enzymeHPRT can be used in conjunction with a vector expressing the hprt gene.The transfected cells are then grown in the presence of hypoxanthine andazaserine (HxAz medium). Examples of dominant selectable markers whichdo not require the use of enzyme-deficient cell lines include the neogene, the hyg gene and the gpt gene.

[0179] When pMSD5-HPRT is used as the selectable marker, the selectivemedium comprises RPMI 1640 medium containing 10% FCS, 100 μMhypoxanthine (Hx) (Sigma) and 2 μg/ml azaserine (Az) (Sigma). Afterapproximately 11 days, positive wells (i.e., wells containing cellscapable of growth in the selective medium) are visible and the coloniesare removed to 24 well plates. The positive colonies are picked from the48 well plates from about day 11 to about 3 weeks following the additionof selective medium.

[0180] Positive colonies removed from the 48 well plates are placed into24 well plates (Costar) in RPMI 1640 medium containing 10% dialyzed FCS(HyClone) and 100 μM Hx. The use of dialyzed serum at this pointincreases the speed and frequency of co-amplification of the input DNAin the transfectants. Hypoxanthine is retained in the culture medium fora few passages until the azaserine is diluted to non-toxicconcentrations.

[0181] The transfected cells which survived growth in selective mediumare then checked to see if they are expressing the genes of interest.This may be done by any suitable assay including cell surface staining,a bioassay for activity, ELISA or immunoprecipitation followed bypolyacrylamide gel electrophoresis. For example if the gene(s) ofinterest encode a cell surface molecule, the transfected cells areanalyzed by staining with an antibody specific for the vector-encodedcell surface molecule. The presence of the antibody on the surface ofthe transfected cell is detected by fluorescence microscopy (thespecific antibody is either directly conjugated to a fluorochrome or afluorescienated secondary antibody is utilized). The best expressingclones are then checked to determine their level of sensitivity to MTX.Typically 6 to 18, more preferably 12, clones are checked.

[0182] The parental (i e., non-transfected) BW5147.G.1.4 cells barelygrow in the presence of 10 nM MTX. By visual inspection 3 to 5 daysafter plating, greater than about 98 percent of the parentalBW5147.G.1.4 cells are killed when 1×10⁴ cells are placed in 2 ml ofmedium containing 20 nM MTX in the well of a 24 well plate (this levelof MTX is referred to as the growth cut off for the parentalBW5147.G.1.4 cell line). At 30 nM MTX, colonies of BW5147.G.1.4 cellsare seen at a frequency of less than 10⁻⁷.

[0183] The transfected and selected cells (“selectants”) are plated in arange of MTX concentrations ranging from 10 to 100 nM; the cells areplated at a density of 1 to 5×10⁴ cells per well in a 24 well plate(Costar); the selectants are plated at the same density of cells as wasused to determine the level of MTX at which > about 98% of the parentalcells were killed. This is done because MTX irreversibly binds to DHFRso that the number of cells present in a given volume effects theconcentration of MTX required to kill the cells; that is if a higherdensity of cell is used, a higher concentration of MTX will be requiredto kill about 98% of the cells [For example when the parental cells areplated at a density of 1×10⁴ cells/2 ml medium in the well of a 24 wellplate 20 nM MTX is sufficient to kill >98% cells in a 3 to 5 day assay.If the density is increased two-fold (1×10⁴ cells in ml medium), 25 nMMTX is required for >98% killing. If 5×10⁴ cells are placed in 2 ml ofmedium in the well of a 24 well plate, 30 nM MTX is required toachieve >98% killing.]

[0184] Clones of selectants typically show growth cut offs of 30 to 60nM MTX (that is greater than about 98% of the selectants are killed whenplaced in medium containing 30 to 60 nM MTX when the plates are visuallyinspected 3 to 5 days after plating in medium containing this level ofMTX). Cells from each selectant of interest which shows MTX resistanceabove the parental BW5147.G.1.4 cells (e.g., above 20 to 30 nM MTX) areplated at 10⁴ cells per well of a 48 well plate (Costar) in 0.5 ml ofRPMI 1640 containing 10% dialyzed FCS and MTX (hereinafter medium-MTX).Several concentrations of MTX are used: 20 nM, 40 nM and 60 nM aboveeach clones' upper level of MTX resistance (i.e., if the upper level ofMTX resistance is 30 nM then the following concentrations may be used:50 nM, 70 nM and 90 nM); these levels of MTX are typically 4-fold to6-fold the level of MTX required to kill greater than about 98% of theparental cells. Any selectants which are capable of growth in mediumcontaining greater than 90 nM MTX are discarded; it has been observedthat selectants which are capable of growing in such high levels of MTXtend to preferentially amplify the amplification vector at the expenseof the expression vector(s).

[0185] After 7 to 10 days, the wells are fed with 0.5 ml medium-MTX.Initial amplificants are picked between 2 to 6 weeks (typically 3 to 5weeks) after plating in medium-MTX. The clones are then analyzed againfor expression of the gene(s) of interest using the appropriate assay(i.e., staining with antibodies for cell surface expression, ELISA,bioassays for activity, immunoprecipitation and PAGE, etc.).

[0186] Typically a HPRT⁺ clone is plated at a concentration of 50 to 80nM MTX (this represent the first round of amplification). The clone isgrown for 2 to 3 weeks and then the level of MTX is increased to 200 nMto 480 nM (a 4 fold increase; this represents the second round ofamplification). After another 2 to 4 weeks, the level of MTX isincreased to 1 to 2 μM MTX (another 4 to 6 fold increase; thisrepresents the third round of amplification). Any clones which show anincreased resistance to MTX without a corresponding increase inexpression of the gene(s) of interest is discarded. Typically anydiscordance is seen on the second round of amplification; such clonesprove to be unstable and are undesirable.

[0187] The methods of the present invention allow, for the first time,the co-amplification of transfected DNA sequences in BW5147 cells. Inaddition, the methods of the present invention provide improved methodsfor the co-amplification of DNA sequences in cell lines. Of theselectants that are expressing the gene(s) of interest, most (i.e.,greater than 80%), if not all, will co-amplify the amplifiable marker(e.g, the dhfr gene which confers resistance to MTX) and the gene(s) ofinterest in the first round of amplification. More than 60% of the firstround amplificants will co-amplify the gene(s) of interest in the secondround in addition to dhfr gene sequences. To date, using the methods ofthe present invention, no clones have been obtained that amplify thegene(s) of interest in the second round of amplification that then failto continue to coordinately amplify in all subsequent rounds until amaximum expression level is reached. Thus, the methods of the presentinvention result in a much higher frequency of coordinateco-amplification of gene sequences than has been reported for othermethods of co-amplification such as that reported by Walls et al. (1989)Gene 81:139 or by Kaufman et al. (1985) Mol. Cell. Biol. 5:1750 whensingle clones were examined. In addition to providing a means forachieving a very high frequency of coordinate co-amplification of genesequences, the methods of the present invention also provide methodswhich produce the desired amplificants with a considerable time savingsrelative to existing methods. The method of the present invention avoidsthe time-consuming step of searching through pools of primarytransformants which have been subjected to a round of amplification tofind those few clones within the pool which are expressing the proteinof interest at high levels.

[0188] The following modifications to the above-described amplificationprotocol have been found to decrease the amount of time required for thefirst round of amplification by 2 to 3 weeks. First, the originaltransfectants are selected by growth in RPMI 1640 medium containing 100μM Hx, 2 μg/ml Az and 10% dialyzed FCS. Second, the originaltransfectants are fed at about 10 days following electroporation with0.5 ml per well (in a 48 well plate) of RPMI 1640 medium containing 10%dialyzed FCS, 100 μM Hx and 10 nm MTX; this yields a final concentrationin each well of the 48 well plate of 5 nM MTX. The net effect of thegrowth of the transfected cells in medium containing dialyzed FCS and 5nM MTX is to give the cells which have undergone amplification events aselective advantage.

[0189] VIII. Co-Amplification Without Prior Selection

[0190] The amplified cell lines of the present invention may begenerated using only an amplification vector in addition to theexpression vector(s) (ie., the use of a selection vector is notrequired). Cell lines which do not lack a functional gene productcorresponding to the enzyme encoded by the amplification vector andwhich can be successfully employed without the use of a selectablemarker in addition to the amplifiable marker are those cell lines inwhich the background level of amplification of the endogenous gene(e.g., the endogenous dhfr gene when DHFR is used as the amplifiablemarker) is low enough that amplification of the input amplifiable gene(ie., the amplification vector) occurs preferentially.

[0191] When it desired that no selection step be employed, the aboveprotocols are modified as follows. The amplification vector andexpression vector(s) are linearized and electroporated into the parentalcell line using a ratio of 1:10-15 (amplification vector:expressionvector). Again large amounts of DNA are introduced, preferably byelectroporation, into the cells. Typically, 20 μg of the amplificationvector is used and 200 to 250 μg each of two expression vectors (or 400to 500 μg of a single expression vector). Following electroporation, thetransfected cells are allowed to recover for 36 to 48 hours as describedabove. The transformed cells are then transferred to 48 well plates at adensity of no more than 1×10⁶ cells per well in medium containing 4-foldto 6-fold the concentration of inhibitor required to prevent the growthof the parental cells. Using the BW5147.G. 1.4 cell line, the expectedfrequency of generating a primary transformant which contains enoughcopies of the input amplifiable gene to allow the isolation of a firstround amplificant capable of growth in medium containing 4- to 6-foldthe level of inhibitor required to prevent growth of the parentalBW5147.G.1.4 cells is approximately 1 in 10⁸ to 1 in 10¹⁰ cells.Accordingly, at least 5×10⁸ to 1×10¹¹ cells must be plated in mediumcontaining elevated levels of the inhibitor to permit the isolation ofseveral first round amplificants. Cells capable of growing in 4- to6-fold the level of inhibitor required to prevent growth of the parentalcells are examined for the ability to express the protein(s) ofinterest; those clones expressing high levels of the protein of interestare subjected to subsequent rounds of amplification as described above.Any clones which do not display a coordinate increase in the level ofexpression of the protein(s) of interest and the level of resistance tothe inhibitor at any amplification step are discarded.

[0192] The ability to generate amplified cell lines without the need toemploy a selection vector reduces the amount of time required toproduced the desired amplified cell line. However, the use of aselection vector and the initial selection step is advantageousparticularly when working with cell lines which have a high backgroundfrequency of amplification of the endogenous locus corresponding to theamplifiable gene present on the amplification vector. Even when workingwith a cell line which does not a have a high background level ofamplification of the endogenous gene, the use of a selection vector andan initial selection step is advantageous because it allows one to workwith only the primary selectants expressing the highest levels of thegene(s) of interest. This reduces the amount of time and effort requiredto generate amplified cell lines expressing very high levels of theprotein(s) of interest.

[0193] IX. High-Level Expression of Interleukin 10 in Amplified CellLines

[0194] Using the methods of the present invention, cell lines wereisolated which express large quantities of interleukin 10 (IL-10). IL-10is a cytokine produced by TH₂ cells (type 2 helper T cells),macrophages/monocytes, and some B cells. IL-10 acts to inhibit thesynthesis of cytokines by activated TH₁ cells, activated macrophages andnatural killer cells [Mosmann (1993) Ann. Rev. Immunol. 11: 165 andMosmann (1994) Advances in Immunol. 56:1]. Studies have shown that IL-10expression is positively correlated with graft outcome intransplantation [Bromberg (1995 Curr. Op. Immunol. 7:639]. Accordingly,there is interest in using IL-10 therapeutically. Therapeutic use ofIL-10, of course, requires the ability to produce large quantities ofIL-10.

[0195] Presently, there are two commercial sources of murine IL-10.Genzyme Diagnostics (Cambridge, Mass.) sells 5 mg of IL-10 produced inE. coli produced for $295.00 (cat#2488-01, ˜2500 units). BiosourceInternational (Camarillo, Calif.) sells 5 mg of IL-10 produced in E.coli for $245.00 (cat# PMC-0104, ˜2500 units). The methods of thepresent invention were used to isolate cell line which produces about75,000 units per milliliter of culture supernatant. Using the lowercommercial price for IL-10, these cells produce about $7,350,000.00worth of IL-10 per liter in a static culture. These amplified cell linesyield about 150 mg of IL-10 protein per liter in static culture; thus,the unpurified culture supernatant from these amplified cell linesprovides a much more pure source of IL-10 than do presently availablecommercial preparations.

[0196] X. High-Level Expression of Human Class II MHC Antigens and TCell Receptor Proteins in Amplified Cell Lines

[0197] The human class II MHC antigens, HLA-DR, and their correspondingmouse analogs, the Ia antigens, are cell surface membrane glycoproteinswhich mediate the recognition of non-self molecules (i.e., antigens) byT lymphocytes. Class II molecules display fragments of foreign antigenson the surface of antigen presenting cells which include macrophages,dendritic cells, B lymphocytes and activated T lymphocytes. WhenMHC-restricted, antigen-specific T lymphocytes interact with antigenpresenting cells bearing class II molecules bound to antigen, an immuneresponse is generated.

[0198] Class II antigens comprise two chains, an α chain and a β chain.Both chains must be expressed in the same cell in order for the class IImolecule to be transported to the surface of the cell. The β chain ishighly polymorphic, and this polymorphism results in heritabledifferences in immune responsiveness. In certain class II MHC molecules(e.g., mouse IA, human DQ and DP), the α chain is also highlypolymorphic. Given the central role that class II molecules play in theimmune response, including rejection of transplanted tissue andheritable susceptibility to auto immune disease, studies of theinteraction of class II molecules with foreign antigen and with Tlymphocytes have been undertaken. These studies of the physical-chemicalinteraction of class II molecules with antigen require the availabilityof large quantities of purified soluble class II molecules. In addition,the use of class II molecules complexed with specific peptides has beensuggested for the treatment of autoimmune disease [Sharma, et al. (1991)Proc. Natl. Acad. Sci. USA 88:11465].

[0199] In order to provide such reagents, chimeric human DR moleculeswere expressed at high levels on the surface of amplified cell linesusing the selection amplification method of the invention. The DRmolecules are, cleaved from the cell surface to produce soluble DRmolecules by treatment with an enzyme capable of cleaving either aphosphatidylinositol linkage or a thrombin site which is present on thechimeric DR molecule.

[0200] A similar approach allows the production of large quantities ofsoluble T cell receptor (TCR) molecules or immunoglobulin (Ig)molecules. Like, class II molecules, TCR and Ig molecules compriseheterodimers (i.e., two different chains associate to form the TCR or Igmolecule displayed on the cell surface; it is noted that both cellsurface and soluble forms of Ig molecules exist in nature and forpatient immunization one would produce soluble Ig). The methods of thepresent invention permit the production of large quantities of solubleforms of class II MHC molecules and TCR to be produced in a rapidmanner. This allowing for the production of customized tumor cellvaccines comprising soluble TCR for the treatment of lymphoma andleukemia patients as well as the production of soluble class II MHCmolecules for the treatment of autoimmune disease.

[0201] XI. Production of Custom Multivalent Vaccines for the Treatmentof Lymphoma and Leukemia

[0202] The existing approach toward vaccination (i.e., activeimmunotherapy) of B-cell lymphoma and leukemia involves the productionof a custom vaccine comprising autologous immunoglobulin idiotype whichcorresponds to the most abundant antibody molecule expressed on thesurface of the B-cell tumor. An analogous approach for the treatment ofT-cell lymphomas and leukemias would involve the production of a customvaccine comprising autologous T cell receptor (TCR) idiotype whichcorresponds to the most abundant TCR molecule expressed on the surfaceof the T-cell tumor.

[0203] It is known in B-cells that the variable regions of rearrangedimmunoglobulin (Ig) genes accumulate point mutations following antigenicstimulation (Ig). This process, known as somatic mutation or somatichypermutation, is responsible for affinity maturation of humoral immuneresponses [Tonegawa (1983) Nature 302:575, Teillaud et al. (1983)Science 222:721, Griffiths et al. (1984) Nature 312:272 and Clarke etal. (1985) J. Exp. Med. 161:687]. During affinity maturation, antibodiesof higher affinity emerge as the immune response proceeds (i.e.,progression from primary to secondary to tertiary responses). Acomparison of the antibody produced during the immune response revealsthat mutations accumulate from the late stage of primary responsesonward; these mutations cluster in the second complementaritydetermining region (CDR2) region of the Ig molecule (ie., within thehypervariable regions within the antigen-binding site). Somatic mutationdoes not occur in T cells.

[0204] Somatic variants are known to exist within the population ofcells comprising certain B-cell tumors (e.g., low grade or follicularB-cell lymphomas); thus, while these tumors are clonal at the level ofIg gene rearrangements (including nucleotide sequence at the V-D-Jjunctions) they are in fact quasi-clonal with respect to the nucleotideor amino acid sequence of their heavy chain V regions [Cleary M L et al.(1986), Cell 44:97 and Levy S et al. (1988) J. Exp. Med. 168:475]. It isthought that following the transformation event(s) which gives rise tothe B-cell tumor, somatic mutation persists. Analysis of B-celllymphomas reveals that about 1 to 5% of the cells within the tumorcontain somatic mutations.

[0205] The fact that somatic variants exist within a B-cell tumor hasimplications for immunotherapy of these tumors. For example, treatmentof B-cell lymphoma with anti-idiotype antibodies was shown to produce aninitial partial response in patients; however, idiotype variant tumorcells (idiotype negative) emerged at the original tumor site [Cleary M Let al. (1986), supra; Bahler D W and Levy R (1992) Proc. Natl. Acad.Sci. USA 89:6770; Zelenetz A D et al. (1992) J. Exp. Med. 176:1137; andZhu D et al. (1994) Brit. J. Haematol. 86:505]. It is thought that theseidiotype variant tumor cells were already present before treatment withthe monoclonal anti-idiotype antibody and that they were allowed toproliferate after the selective removal of the idiotype positive tumorcells. These clinical trials showed the drawback of targeting a singleepitope on the tumor cell.

[0206] In order to improve the immunotherapy of B-cell tumors, activeimmunization with autologous tumor derived Ig or Ig subfragments hasbeen employed. It is hoped that the use of the Ig or Ig subfragments asan immunogen would produce a T cell response and antibodies directedagainst a number of different epitopes found within the tumor-specificIg. In this type of treatment the Ig (or idiotype fragment of the Ig) ofthe patient's tumor cell is expressed While this approach has theadvantage that multiple epitopes are targeted, it still suffers from thefact that a single Ig (or subfragment) is used as the immunogen andtherefore the possibility exists that tumor cells expressing somaticvariants of the predominant Ig will escape and proliferate. To producethe tumor Ig-idiotype protein used for immunization with existingmethodologies, lymphoma cells removed by surgical biopsy are fused withthe heterohybridoma cell line K6H6/B5 which has lost the ability tosecrete endogenous Ig. Hybrid cells which secrete Ig corresponding tothe immunophenotype of the tumor sample are expanded and the secreted Igis purified for use as a vaccine [Kwak et al. (1992), supra]. Thistechnique is referred to as “rescue fusion.” The Ig produced by rescuefusion represents a single Ig derived from the patient's tumor; this Igis presumably the predominant Ig expressed by the tumor. Thus, vaccinesproduced by rescue fusion are monovalent and do not represent the fullcomplexity of Ig expressed by tumors which contain somatic variants.

[0207] Clinical trials using tumor Ig-idiotype protein produced byrescue fusion to vaccinate B-cell lymphoma patients are in progress.These trials are showing impressive clinical improvements for thesetumors which remain essentially incurable with conventional therapy(i.e., chemotherapy). This custom vaccine therapy is used following acourse of conventional chemotherapy (employed to reduce the tumorburden). The clinical improvements are seen when comparing patientstreated solely with conventional chemotherapy with patients who receivedcustom vaccines following chemotherapy. Among the patients who have beentreated with custom vaccines and followed for a lengthy period of time(about 8 years), one has recently relapsed. Although not confirmed atthis time, it is possible that this relapse is due to the outgrowth oftumor cells bearing somatic variants of the tumor Ig-idiotype proteinused in the vaccine.

[0208] In addition to the failure to provide a multivalent vaccinerepresentative of all Ig variants present in the patients tumor, therescue fusion technique has other draw backs. This technique requires alarge number of tumor cells which are obtained by surgical biopsy ofenlarged lymph nodes in the patient. Some B-cell lymphoma patients donot present with enlarged lymph nodes suitable for surgical biopsy andtherefore cannot be treated using vaccines produced by the rescue fusiontechnique. Furthermore, the production of each custom heterohybridomacell line secreting the patients Ig takes between 2 to 8 months (averageof 4 months) and is labor intensive; in laboratories having extensiveexperience with the rescue fusion technique, the rate of vaccineproduction is about 25 custom vaccines per technician per year. Thisrate of producing custom vaccines is not sufficient to meet the existingand growing patient demand.

[0209] Ideally, the method for producing custom tumor specific vaccinescould be performed on a small number of cells (i.e., from a fme needlebiopsy), would produce a multivalent vaccine representative of the fullcomplexity of the patient's tumor, would be fast (average of 2-3 months)and would be less labor intensive than existing methods such that asingle technician could produce at least a hundred custom vaccines peryear.

[0210] The methods described herein (Examples 9 and 10) provide a meansto produce custom vaccines, including multivalent vaccines, from smallnumbers of cells quickly and efficiently. The ability to use a smallsample size permits the production of custom vaccines for patientslacking enlarged lymph nodes suitable for surgical biopsy. In additionto expanding the pool of patients who can be treated with customvaccines, the ability to use fine needle biopsy material obviates theneed for surgery for all lymphoma patients (at least with respect to thecollection of cells for the production of custom vaccines).

EXPERIMENTAL

[0211] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0212] In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM(nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg(picograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); AMP (adenosine 5′-monophosphate); cDNA (copy orcomplimentary DNA); DNA (deoxyribonucleic acid); ssDNA (single strandedDNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline);g (gravity); OD (optical density); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymeraseI large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethyleneglycol-bis(β-aminoethyl ether) N, N, N′, N′-tetraacetic acid); EDTA(ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); ORI (plasmid origin of replication); lacI(lac repressor); Amicon (Amicon Corp., Beverly, Mass.); ATCC (AmericanType Culture Collection, Rockville, Md.); Becton Dickinson (BectonDickinson Immunocytometry Division, San Jose Calif.); Costar (Costar,Cambridge, Mass.); Falcon (division of Becton Dickinson Labware, LincolnPark, N.J.); FMC (FMC Bioproducts, Rockland, Me.); Gibco/BRL (Gibco/BRL,Grand Island, N.Y.); HyClone (HyClone, Logan, Utah); Sigma (SigmaChemical Co., St. Louis, Mo.); NEB (New England Biolabs, Inc., Beverly,Mass.); Operon (Operon Technologies, Alameda, Calif.); Perkin-Elmer(Perkin-Elmer, Norwalk, Conn.); Pharmacia (Pharmacia Biotech,Pisacataway, N.J.); Promega (Promega Corp., Madison, Wis.); Sarstedt(Sarstedt, Newton, N.C.); Stratagene (Stratagene, LaJolla, Calif.); U.S.Biochemicals (United States Biochemical, Cleveland, Ohio); and Vector(Vector Laboratories, Burlingame, Calif.).

EXAMPLE 1 Construction of Expression Vectors

[0213] In order to construct the expression vectors of the invention anumber of intermediate vectors were first constructed.

[0214] Construction of pSSD5 and pSSD7

[0215] pSSD5 and pSSD7 contain the following elements from SV40: theenhancer/promoter region, the 16S splice donor and acceptor and the polyA site. Vectors containing the SV40 enhancer/promoter sequences willreplicate extrachromosomally in cell lines which express the SV40 largeT antigen as the SV40 enhancer/promoter sequences contain the SV40origin of replication.

[0216] A polylinker containing the recognition sequences for severalrestriction enzymes is located between the splice acceptor and poly Asequences. The polylinker allows for the easy insertion of a gene ofinterest. The gene of interest will be under the transcriptional controlof the SV40 enhancer/promotor. pSSD5 and pSSD7 differ only in thesequences of the polylinker (sequences listed below). The polylinker ofpSSD5 contains the following restriction sites: XbaI, NotI, SfiI, SacIIand EcoRI. The polylinker of pSSD7 contains the following restrictionsites: XbaI, EcoRI, MluI, StuI, SacII, SfiI, NotI, BssHII and SphI.

[0217] pSSD5 was constructed by digestion of the plasmid pL1 [Okayamaand Berg, Mol. Cell. Biol., 3:280 (1983)] with PstI and HindIII. Allrestriction enzymes were obtained from New England Biolabs and were usedaccording to the manufacturer's directions. The plasmid pcDV1 [Okayamaand Berg, supra] was digested with HindIII and BamHI. Both digests wereelectrophoresed on a 0.8% low melting temperature agarose gel(SeaPlaque, FMC). A 535 bp DNA fragment from the pLI digest containingthe SV40 enhancer/promoter and 16S splice junctions was cut out of thegel. A 2.57 kb DNA fragment from the pcDVl digest containing the SV40polyadenylation signals and the pBR322 backbone was cut out of the gel.The two gel slices were combined in a microcentrifuge tube and theagarose was removed by digestion with β-Agarase I (NEB) followed byisopropanol precipitation according to the manufacturer's directions.The DNA pellets were dried and resuspended in 20 μl of TE.

[0218] Two synthetic oligonucleotides (Operon), SD5A [5′-TCTAGAGCGGCCGCGGAGGCCGAATTCG-3′ (SEQ ID NO:1)] and SD5B [5′-GATCCGAATTCGGCCTCCGCGGCCGCTCTAGATGCA-3′ (SEQ ID NO:2)] were added in equal molar amountsto the resuspended DNA fragments. Ligation buffer (10X concentrate, NEB)was added to a 1X concentration, 80 units of T4 DNA ligase was added andthe ligation was placed at 14° C. overnight. Competent E. coli cellswere transformed with the ligation mixture and a plasmid was isolatedthat consisted of the DNA fragments from pL1 and pcDV1 with a novelpolylinker connecting the fragments. The resulting plasmid was namedpSSD.

[0219] The 670 bp BamHI/PstI fragment containing the SV40 poly Asequences (SV40 map units 2533 to 3204; SEQ ID NO:3) was removed fromSV40 DNA and cloned into pUC19 digested with BamHI and PstI. Theresulting plasmid was then digested with BclI (corresponds to SV40 mapunit 2770). The ends were treated with the Klenow enzyme (NEB) and dNTPsto create blunt ends. Unphosphorylated PvuI1 linkers (NEB) were ligatedto the blunted ends and the plasmid was circularized to create pUCSSD.The SV40 poly A sequences can be removed from pUCSSD as a BamHI/PvuIIfragment.

[0220] pSSD5 was constructed by ligating together the following threefragments: 1) the 1873 bp SspI/PvuII fragment from pUC19; this providesthe plasmid backbone; 2) the 562 bp fragment containing the SV40enhancer/promoter and 16S splice junction and the polylinker from pSSD;this fragment was obtained by digestion of pSSD with SspI and partialdigestion with BamHI followed by isolation on low melting agarose andrecovery as described above; and 3) the 245 bp BamHII/PvuII fragmentfrom pUCSSD (this fragment contains the SV40 poly A sequences). Thethree fragments were mixed together and ligated using T4 DNA ligase(NEB) to create pSSD5. The map of pSSD5 is shown in FIG. 1.

[0221] To create pSSD7, pSSD5 was digested with XbaI and EcoRI. Thesynthetic oligonucleotide pair SD7A and SD7B (Operon) was ligated intothe cut pSSD5 thereby generating the SD7 polylinker. The sequence ofSD7A is 5′-CTAGAATTC ACGCGTAGGCCTCCGCGGCCGCGCGCATGC-3′ (SEQ ID NO:4).The sequence of SD7B is 5′-AATTGCATGCGCGCGGCCGCGGAGGCCTACGCGTGA ATT-3′(SEQ ID NO:5). The map of pSSD7 is shown in FIG. 2.

[0222] Construction of pSRαSD5 and pSRαSD7

[0223] pSRαSD5 and pSRαSD7 contain the SRa enhancer/promoter followed bythe 16S splice junction of SV40 and either the polylinker formed by theoligonucleotide pair SD5A/SD5B or SD7A/SD7B. The polylinker is followedby the SV40 poly A sequences. A gene of interest can be inserted intothe polylinker and the expression of the inserted gene will be under thecontrol of the human SRα enhancer/promoter. The SRα enhancer/promoters ahybrid enhancer/promoter comprising human T cell leukemia virus 1 5′untranslated sequences and the SV40 enhancer [Takebe et al., Mol. Cell.Biol., 8:466 (1988)]. The SRα enhancer/promoter is reported to increaseexpression from the SV40 enhancer/promoter by ten-fold in host cells.This enhancer/promoter is active in a broad range of cell types. Vectorscontaining the SRα enhancer/promoter will replicate in cells expressingSV40 large T antigen as the SV40 origin of replication is present withinthe SRα enhancer/promoter sequences.

[0224] The SRα enhancer/promoter was removed from pcDL-SRα296 bydigestion with HindIII and XhoI. The ˜640 bp HindIII/XhoI fragment (SEQID NO:6) was recovered from a low melting agarose gel as describedabove. This ˜640 bp fragment was inserted into either pSSD5 or pSSD7digested with HindIII and XhoI (removes the SV40 enhancer/promoter frompSSD5 or pSSD7). The map of pSRαSD5 is shown in FIG. 3. The map ofpSRαSD7 is shown in FIG. 4.

[0225] Construction of pMSD5 and pMSD7

[0226] pMSD5 and pMSD7 contain the long terminal repeat (LTR) from theMoloney murine leukemia virus followed by the 16S splice junction ofSV40 and either the polylinker formed by the oligonucleotide pairSD5A/SD5B or SD7A/SD7B. The polylinker is followed by the SV40 poly Asequences. A gene can be inserted into the polylinker and the expressionof the inserted gene will be under the control of the Moloney LTR.

[0227] The Moloney LTR was removed from a plasmid containing Moloneymurine leukemia viral DNA [Shinnick et al., Nature 293:543 (1981)] bydigestion of the plasmid with ClaI (corresponds to Moloney map unit7674). The ends were made blunt by incubation with Klenow and dNTPs.Unphosphorylated HindIII linkers (NEB) were ligated onto the blunt ends.This treatment destroyed the ClaI site and replaced it with a HindIIIsite. The plasmid was then digested with SmaI (corresponds to Moloneymap unit 8292) and unphosphorylated XhoI linkers were ligated onto theends. The resulting plasmid now contains a XhoI site replacing the SmaIsite at Moloney map unit 8292 and a HindIII site replacing the ClaI siteat Moloney map unit 7674. The plasmid was then digested with XhoI andHindIII. The resulting XhoI/HindIII fragment containing the Moloney LTR(SEQ ID NO:7) was inserted into pSSD5 digested with XhoI and HindIII(this removes the SV40 enhancer/promoter and 16S splice junction frompSSD5) to yield pMSD5. The map of pMSD5 is shown in FIG. 5.

[0228] To create pMSD7, the Moloney LTR on the XhoI/HindIII fragment wasinserted into pSSD7 digested with XhoI and HindIII. The map of pMSD7 isshown in FIG. 6.

[0229] Construction of Vectors Containing the Human Elongation Factor 1αEnhancer/Promoter

[0230] The human elongation factor 1α enhancer/promoter is abundantlytranscribed in a very broad range of cell types. Vectors containing twoversions of this active enhancer/promoter were constructed: 1) a longversion containing ˜1.45 kb of sequences located upstream of theinitiation codon and continuing through the first intron to provide asplice junction and 2) a short version containing 475 bp of sequencesupstream of the initiation codon. The short version of the promoter istermed the “A” version and the long version is termed the “B” version.

[0231] A. Construction of pHEF1αASD5 and pHEF1αASD7

[0232] pHEF1αASD5 and pHEF1αASD7 contain the short version of the humanelongation factor 1α enhancer/promoter [Uetsuki et al., J. Biol. Chem.,264:5791 (1989) and Mizushima and Nagata, Nuc. Acids. Res., 18:5322(1990)]. The human elongation factor 1α enhancer/promoter is abundantlytranscribed in a very broad range of cell types including L929, HeLa,CHU-2 and COS cells.

[0233] The human elongation factor 1α enhancer/promoter (nucleotides 125to 600 of the human elongation factor 1α gene; SEQ ID NO:8) was isolatedfrom human genomic DNA as follows. Genomic DNA was isolated from the MOUcell line (GM 08605, NIGMS Human Genetic Mutant Cell Repository, Camden,N.J.) using standard techniques [Sambrook et al, supra at pp.9.16-9.23]. The MOU cell line is an Epstein-Barr virus transformed humanB cell line.

[0234] Two synthetic oligonucleotide primers (Operon) were used to primethe polymerase chain reaction (PCR) for the isolation of an ˜475 bpfragment containing the human elongation factor 1α enhancer/promoter(SEQ ID NO:8). U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188 coverPCR methodology and are incorporated herein by reference.

[0235] The 5′ primer, designated HEF1αL5, contains the followingsequence: 5′-AAGCTTTGGAGCTAAGCCAGCAAT-3′ (SEQ ID NO:9). The 3′ primer,designated HEF1αL3A, contains the following sequence: 5′-CTCGAGGCGGCAAACCCGTTGCG-3′ (SEQ ID NO:10). PCR conditions were as reported in Saikiet al., Science 239:487 (1988). Briefly, 10 μg MOU genomic DNA and 1 μMfinal concentration of each primer were used in a 400 μl PCR reaction.Reaction conditions were 94° C. for 1 minute, 60° C. for 1 minute, 72°C. for 1.5 minutes, 30 cycles. Taq DNA polymerase was obtained fromPerkin-Elmer. The primer pair generates a 475 bp fragment having aHindIII site at the 5′ end and a XhoI site at the 3′end. The PCRreaction products were electrophoresed on a low melting agarose gel andthe 475 bp fragment was recovered as described above. The recoveredfragment was digested with HindIII and XhoI and inserted into eitherpSSD5 or pSSD7 digested with HindIII and XhoI to yield pHEF1αASD5 andpHEF1αASD7, respectively. The maps of pHEF1αASD5 and pHEF1αASD7 areshown in FIG. 7 and 8, respectively.

[0236] B. Construction of pHEF1αBSD5 and pHEF1αBSD7

[0237] pHEF1αBSD5 and pHEF1αBSD7 were constructed as described above forpHEF1αASD5 and pHEF1αASD7 with the exception that the HEF1αL3B primerwas used instead of the HEF1αL3A primer with the HEF1αL5 primer togenerate a ˜1.45 kb fragment containing the human elongation factor laenhancer/promoter and a splice donor and acceptor from the humanelongation factor 1α gene. The ˜1.45 kb fragment corresponds to mapunits 125 to 1567 in the human elongation factor 1α gene (SEQ ID NO:11).The sequence of HEF1αL3B is 5′-TCTAGAGTTTTCACG ACACCTGA-3′ (SEQ IDNO:12). The HEF1αL3B primer generates a XbaI site at the 3′ end of the˜1.45 kb fragment. This fragment was digested with HindIII and XbaI andinserted into either pSSD5 or pSSD7 digested with HindIII and XbaI togenerate pHEF1αBSD5 or pHEF1αBSD7, respectively. Digestion of pSSD5 andpSSD7 with HindIII and XbaI removes the SV40 enhancer/promoter and theSV40 16S splice junction. These SV40 sequences are replaced with thehuman elongation factor 1α enhancer/promoter and a splice donor andacceptor from the human elongation factor 1α gene. The maps ofpHEF1αBSD5 and pHEF1αBSD7 are shown in FIGS. 9 and 10, respectively.

EXAMPLE 2 Construction of the Selection Vector pMSD5-HPRT

[0238] pMSD5-HPRT contains a full length cDNA clone encoding the mouseHPRT enzyme under the transcriptional control of the Moloney LTR. TheMoloney LTR contains a strong enhancer/promoter which is active in abroad range of cell types [Laimins et al., Proc. Natl. Acad. Sci. USA79:6453 (1984)]. The pMSD5-HPRT expression vector is used as theselective plasmid (or selective or selectable marker) when HPRT⁻ celllines, such as BW5147.G. 1.A, are used as the recipient cell line forthe generation of stable transformants. HPRT⁻ cell lines cannot grow inmedium containing hypoxanthine, aminopterin or azaserine and thymidine(HAT medium). The addition of a functional HPRT gene by gene transferallows the cells which have integrated the vector DNA encoding the HPRTgene to grow in HAT medium.

[0239] a. Isolation Of A Full Length Mouse HPRT cDNA

[0240] A cDNA library was prepared from poly A⁺ mRNA isolated from C6VLcells [Allison et al., J. Immunol., 129:2293 (1982)] using standardtechniques [Sambrook et al., supra at 7.26-7.29]. cDNA was generatedfrom the mRNA and inserted into the expression vector λgt10 usingstandard techniques [Huynh, et al., in DNA Cloning: A Practical Approach(D. M. Glover, ed.), Vol. 1, IRL Press Oxford (1985), pp. 49-78]. Thefull-length mouse HPRT cDNA was isolated using a full-length human HPRTcDNA clone containing an approximately 1.4 kb PstI-BamHI restrictionfragment as a probe [pcD-HPRT; Jolly et al. (1983) Proc. Natl. Acad.Sci. USA 80:477]. The full length cDNA clone was digested with NotI andEcoRI to generate a 1.3 kb fragment containing the coding region of HPRT(the coding region of the mouse HPRT is listed in SEQ ID NO:13; theamino acid sequence encoded within SEQ ID NO:13 is listed in SEQ IDNO:14).

[0241] pMSD5 (described in Example 1) was digested with NotI and EcoRIand the 1.3 kb NotI/EcoRI fragment containing the mouse HPRT cDNA wasinserted to generate pMSD5-HPRT. The map of pMSD5-HPRT is shown in FIG.11.

EXAMPLE 3 Construction of the Amplification Vector pSSD7-DHFR

[0242] pSSD7-DHFR contains a full length copy of the mouse DHFR cDNAunder the transcriptional control of the SV40 enhancer/promoter. Thispromoter/enhancer is active in a wide variety of cell types from manymammalian species [Dijkema et al., EMBO J., 4:761 (1985)]. pSSD7-DHFR isreferred to as the amplifiable marker as the use of this vector allowsthe selection of cell lines which have amplified the vector sequences byselecting for cell which can grow in increasing concentrations of MTX.

[0243] The mouse DHFR cDNA was isolated from double stranded cDNAgenerated from liver RNA using the PCR as follows. Poly A⁺ RNA wasisolated from the liver of (Balb/c×C57B1/6) F1 mice using standardtechniques. First strand cDNA was synthesized from the poly A⁺ RNA in afinal reaction volume of 100 μl. The following reagents were added inorder: 35.6 μl H₂O, 5 μl poly A⁺ RNA (1 μg) and 1.4 μl SBNSSdT primer (1μg). The sequence of the SBNSSdT primer is 5′-GCATGCGCGCGGCCGCGGAGGCTTTTTTTTTTTTTTTTTT-3′ (SEQ ID NO:15). The water,primer and RNA were heated at 60° C. for 2 minutes then placed on ice.Forty μl of all four dNTPs at 5 mM each, 10 μl 10X reverse transcriptasesalts (1.0 M Tris-HCl, pH 8.3, 0.5 M KCl, 0.1 M MgCl₂, 0.1 M DTT), 2 μlRNasin (Promega) and 5 μl AMV reverse transcriptase (Molecular GeneticResources, Tampa, Fla.). The reaction was run at 41° C. for 3 hours. Thereaction was stopped by incubation at 65° C. for 10 minutes.

[0244] The reaction components were transferred to a Centricon 100 tube(Amicon) and 2.1 ml of 5 mM Tris-HCl, pH 8.3 was added. The tube wascentrifuged at 300 rpm (˜700 g) for 4 minutes at 10° C. 2.2 ml ofTris-HCl, pH 8.3 was added and the tube was centrifuged again as above.This washing step was repeated and then the tube was inverted andcentrifuged at 2500 rpm for 5 minutes at 10° C. to recover the firststrand cDNA (volume ˜50 μl). Second strand cDNA was synthesized asfollows. 96 μl H₂O and 20 μl 10X rTth RTase buffer (900 mM KCl, 100 mMTris-HCl, pH 8.3) was added to the first strand cDNA. In a separate tubethe following components were mixed: 20 μl 10 mM MnCl₂, 4 μl of each ofthe four dNTPs at 10 mM and 10 μl rTth reverse transcriptase(Perkin-Elmer). Both mixtures were heated to 60° C. and the secondmixture was added to the cDNA mixture. The reaction was carried out at60° C. for 10 minutes. The reaction was stopped by addition of 25 μlchelating buffer [50% glycerol (v/v), 1 mM KCl, 100 mM Tris-HCl, pH 8.3,7.5 mM EGTA, 0.5% Tween 20] and the mixture was placed on ice.

[0245] The reaction mixture was then transferred to a Centricon 100 tubeand 2.1 ml of 5 mM Tris-HCl, pH 7.5 was added. The tube was centrifugedat 5500 rpm for 30 minutes at 10° C. 2.2 ml of Tris-HCl, pH 7.5 wasadded and the tube was centrifuged again as above. This washing step wasrepeated and then the tube was inverted and centrifuged at 2500 rpm for5 minutes at 10° C. to recover the double stranded cDNA (volume ˜50 μl).The cDNA was precipitated with ethanol, resuspended in sterile H₂O andquantitated by absorption at 260 and 280 nm.

[0246] Two hundred pg of double stranded cDNA was used in a 400 μl PCRreaction. The primer set used to prime the PCR was: muDHFR.A: 5′-CGGCAACGCGTGCCATCATGGTTCGAC-3′ (SEQ ID NO:16) and muDHFR.B: 5′-CGGCAGCGGCCGCATAGATCTAAAGCCAGC-3′ (SEQ ID NO:17). The PCR reaction conditionswere as reported in Saiki et al., Science 239:487 (1988). Briefly, thereaction was run at 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for1.5 minutes and 30 cycles were performed. Taq DNA polymerase wasobtained from Perkin-Elmer and the reaction buffer used was thatrecommended by the manufacturer. The primer pair generates a 671 bpfragment having a MluI site at the 5′ end and a NotI site at the 3′ end(SEQ ID NO:18; the amino acid sequence encoded by SEQ ID NO:18 is listedin SEQ ID NO:19). The PCR reaction products were digested with MfluI andNotI and electrophoresed on a low melting temperature agarose gel(SeaPlaque, FMC). The 671 bp fragment was cut out of the gel and theagarose was removed by digestion with β-Agarase I (NEB) followed byisopropanol precipitation according to the manufacturer's directions.

[0247] The 671 bp fragment was inserted into pSSD7 which was digestedwith MluI and NotI to generate pSSD7-DHFR. The map of pSSD7-DHFR isshown in FIG. 12.

EXAMPLE 4 Construction of the Expression Vector pJFE 14ΔIL10

[0248] pJFE 14ΔIL10 contains a full length cDNA clone encoding the mouseinterleukin 10 (IL-10) protein under the transcriptional control of theSRα enhancer/promoter. As discussed above, the SRα enhancer/promoter isactive in a broad range of cell types. pJFE 14ΔIL10 is used to directthe expression of the IL-10 gene in transfected cells (i.e., pJFE14ΔIL10 expresses IL-10 as the gene of interest).

[0249] a. Construction Of pJFE 14ΔIL10

[0250] The plasmid pJFE14 [Elliott et al. (1990) Proc. Natl. Acad. SciUSA 87:6363] was constructed by combining DNA fragments from theplasmids pSSD, pcDL-SRα296 [Takebe et al. (1988) Mol. Cell. Biol. 8:466]and pCDM8 [Seed (1987) Nature 329:840]. pSSD was cut with HindIII andXhoI and a 2.77 kb fragment was isolated from an agarose gel. pcD-SRα296was cut with HindIII and XhoI and an ˜640 bp fragment was isolated froman agarose gel. The two gel-purified DNA fragments were ligated togetherto generate the plasmid pSRαSD. pSRαSD was cut with XbaI and NotI and a3.4 kb fragment was isolated from an agarose gel. pCMD8 was cut withXbaI and NotI and a 440 bp fragment was isolated. The 3.4 kb and 440 bpXbaI/NotI fragments were ligated together to generate pJEL14. Aschematic of pJFE14 is shown in FIG. 13.

[0251] The ΔIL10 cDNA was generated from a full-length mouse cDNA clone,F115 [Moore et al. (1990) Science 248:1230] using the PCR. ThepcDSRα-F115 clone was linearized with BamHI, which cuts out the cDNAinsert. A PCR reaction was run using AmpliTaq™ DNA Polymerase (PerkinElmer) and buffer supplied by the manufacturer according to theirsuggested conditions. The primers used in the PCR were IL10Δ-5′[5′-ATATATCTAGACCACCATGCCTGGCTCAGCACTG-3′ (SEQ ID NO:20)] andIL10Δ-3′[5′-ATTATTGCGGCCGCTTAGCTTTTCATTTTGAT CAT-3′ (SEQ ID NO:21)]. ThePCR reaction was run at 94° C., 1 min, 72° C., 1 min, 46° C., 1 min for30 cycles. The PCR generated DNA has deleted essentially all of the non-coding sequences and placed an optimal Kozak sequence just 5′ to theinitiator ATG of the IL-10 gene sequences. The PCR generated DNA wasextracted with phenol:CHCl₃ (1:1) and then with CHCl₃. The DNA wasethanol precipitated, pelleted in a microcentrifuge and resuspended inTE. The DNA was cut with XbaI and NotI. pJFE14 was cut with XbaI andNotI. Both digestion mixtures were run on a low melt agarose gel. The550 bp ΔIL10 band and the 3.4 kb pJFE14 band were cut out of the gel andcombined in a tube. The DNAs were co-extracted from the agarose, ligatedtogether and transformed into the bacteria DH5α. Colonies were pickedand the clone pJFE14-ΔIL10 was identified. A schematic map ofpJFE14-ΔIL10 is shown in FIG. 14.

EXAMPLE 5 Construction of pSRαSD5-DRα-DAF

[0252] pSRαSD5-DRα-DAF contains a cDNA clone encoding a chimeric mouseDRα gene. In this chimeric protein, the extracellular domain of the DRαprotein is joined to sequences derived from the decay acceleratingfactor (DAF) gene. The DAF sequences provide a glycophosphatidylinositollinkage which allows the chimeric protein to be cleaved from the surfaceof the cell (cell surface expression requires the expression of the DRβchain in the same cell) by treatment of the cell with phospholipase C.

[0253] a. Construction Of The Phagemid Vector pDAF20

[0254] To generate pSRαSD5-DRα-DAF and pSRαSD5-DRβ1-DAF (Example 6), avector containing sequences encoding a portion of decay acceleratingfactor (DAF) which anchors DAF to the cell surface via aglycophosphatidylinositol linkage was constructed. pDAF20 wasconstructed as follows.

[0255] Two micrograms of pBluescript KS(−) (Stratagene) was cut withEcoRV (NEB). TE buffer was added to such that the final volume was 200μl. Spermine was added to a final concentration of 1.4 mM and the DNAwas allowed to precipitate for 20 minutes on ice. The precipitated DNAwas then pelleted by centrifugation for 10 min. in a microcentrifuge andthe spermine was washed from the pellet exactly as described [Hoopes andMcClure (1988) Nucleic Acids Res. 9:5493]. Briefly, the pellet wasdispersed in extraction buffer [75% EtOH, 1X Buffer 2 (0.3M sodiumacetate, 0.01M magnesium acetate)] by vortexing; the dispersed pelletwas then left on ice for 1 hour. The pellet was collected bycentrifugation for 10 min. in a microcentrifuge. The pellet was dried atroom temperature and resuspended in 14 μl H₂O. On ice, 250 ng each ofDAFa (SEQ ID NO:22) and DAFb (SEQ ID NO:23) unphosphorylatedoligonucleotides were added to the resuspended DNA. TheDNA-oligonucleotide mixture was then brought to a final concentration of50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT and 1 mM rATP in a finalreaction volume of 20 μl. Eighty units of T4 DNA ligase (NEB) was addedand the ligation mixture was placed at 14° C. overnight. The ligationmixture was then heated to 65° C. for 10 min. NaCl was added to a finalconcentration of 50 mM and the DNA was digested with EcoRV (NEB). Analiquot of the DNA was then used to transform competent HB101.

[0256] Clones were picked and miniprep DNA was examined by restrictionenzyme digestion. A clone, called DAF20, was isolated that has the DAFsequence cloned in the EcoRV site of pBluescript KS(−) with the XbaI atone end of the DAF sequence adjacent to the EcoRI site in the polylinkerand away from the HindIII site in the polylinker. The sequence of thepDAF20 polylinker region containing the DAF insert is listed in SEQ IDNO:24.

[0257] The resulting plasmid pDAF20 contains DNA encoding the final 37amino acids of the form of DAF that is anchored to the cell surface by aglycophosphatidylinositol (PI) linkage [Caras et al. (1987) Nature325:545]. Chimeric proteins containing these 37 amino acids at theirC-terminus, can be expressed on the cell surface of mammalian (andinsect) cells with this PI anchor. This anchor can be readily cleavedand the protein solubilized from the cell surface usingphosphatidylinositol-specific phospholipase C [Caras et al. (1987)Science 238:1280].

[0258] Phosphatidylinositol-specific phospholipase C was purified fromBacillus thuringiensis (ATCC 10792) exactly as described [Kupke et al.(1989) Eur. J.

[0259] Biochem. 185:151]; phosphatidylinositol-specific phospholipase Cis available commercially (e.g., Sigma).

[0260] The use of soluble class II molecules complexed with specificpeptides has been suggested for the treatment of autoimmune disease[Sharma, et al. (1991) Proc. Natl. Acad. Sci. USA 88:11465]. Suchtherapy requires that ample quantities of soluble class II molecules beavailable. The present invention allows large quantities of solubleclass II molecules to be produced from cells expressing class IImolecules on the cell surface wherein these molecules are anchored tothe cell via the PI anchor provided by sequences derived from DAF.Alternatively, soluble forms of cell surface proteins can be producedaccording to the methods of the present invention using DNA sequencesencoding chimeric class II molecules containing a thrombin cleavage sitebetween the extracellular domain and the transmembrane domain of eachchain comprising the class II heterodimer.

[0261] b. Isolation Of A Full-Length HLA DRα cDNA

[0262] A cDNA library was prepared from poly A⁺ mRNA isolated from IBw4cells (GM03104B, NIGMS Human Genetic Mutant Cell Repository at theCoriell Institute for Medical Research, Camden, N.J.) using standardtechniques [Sambrook et al., supra at 7.26-7.29]. cDNA was generatedfrom the mRNA and inserted into the cloning vector λgt10 using standardtechniques [Huynh et al., in DNA Cloning: A Practical Approach (D. M.Glover, ed.), vol. 1, IRL Press Oxford (1985), pp. 49-78]. A full-lengthDRα cDNA was isolated from the library using a partial DRα cDNA as aprobe; the partial DRα cDNA was contained within pDRα1 [Stetler et al.(1982) Proc. Natl. Acad. Sci. USA 79:5966]. The resulting full-lengthDRα cDNA was contained on a 1.2 kb NotI/EcoRI fragment.

[0263] c. Construction Of SRαSD5-DRα-DAF

[0264] An in-frame connection between the extracellular coding sequenceof DRα and the DAF sequence was performed using site-directed in vitrodeletional mutagenesis [Kunkel et al. (1987) Methods in Enzymology154:367]. The mutational, bridging oligonucleotide encodes the desiredconnection.

[0265] The full length DRα cDNA was subcloned as a NotI-EcoRI fragmentinto pDAF20 (section a above). The pDAF20-DRα was isolated andtransformed into the bacteria BW313 [Kunkel et al. (1987), supra]. Acolony was then grown overnight in LB containing 100 μg/ml ampicillin.The overnight culture was diluted 1:10 in a final volume of 6 ml andgrown at 37° C. After 1 hour, 400 μl of a stock of helper phage R408[Russel et al. (1986) Gene 45:333] having a titer of approximately1×10¹¹ pfu/ml was added to the culture and the culture was grown at 37°C. for approximately 8 hours. One point four (1.4) ml aliquots of theculture were then placed into 4 microcentrifuge tubes and spun in amicrocentrifuge 5 min at 4° C. One point one (1.1) ml of eachsupernatant was transferred to fresh microcentrifuge tubes containing150 μl of 20% PEG(6000), 2.5 M NaCl. The contents of the tubes weremixed and allowed to stand at room temp. for at least 20 min.Precipitated, ssDNA containing phage particles were pelleted in amicrocentrifuge for 5 min at 4° C. Care was taken to remove all thePEG-containing supernatant from the pellets. The four pellets wereresuspended in a total of 200 μl of 300 mM NaOAc, pH 7 and extractedwith an equal volume of phenol:CHCl₃ (1:1) twice, and then once withCHCl₃. Two volumes of ethanol was added to the supernatant and chilledto −20° C. The ssDNA was pelleted in a microcentrifuge 20 min at 4° C.The pellet was dried and resuspended in 10 μl TE buffer.

[0266] The bridging oligonucleotide was phosphorylated in a volume of 20μl containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 10 mM DTT, 1 mM rATPand 65 ng of the RADAF2 oligonucleotide (SEQ ID NO:25) with 8 units ofT4 DNA polynucleotide kinase (Phannacia) at 37° C. for 1 hour. To annealthe bridging oligonucleotide to the ssDNA template, 1.1 μl of thephosphorylated RADAF2 oligonucleotide (SEQ ID NO:25) and 5 μl of thessDNA prep were mixed in a final volume of 15 μl of 40 mM Tris-HCl (pH7.5), 20 mM MgCl₂, 50 mM NaCl, heated to 70° C. and allowed to cool toroom temp. on the bench top. In the reaction tube, the concentrations ofthe buffers were adjusted to give, in a final volume of 95 μl, 16.8 mMTris-HCl, pH 7.5, 11.6 mM MgCl₂, 7.9 mM NaCl, 10.5 mM DTT and 1.1 mMrATP. Four units of T4 DNA ligase (NEB) and 3.8 units of Sequenase (USBiochemicals) were added to the reaction, which was incubated at roomtemp. for 5 min and 37° C. for 1 hour. The reaction was adjusted to 58mM NaCl and heated at 65° C. for 10 min. The tube was cooled to 37° C.and the DNA cut with EcoRI and XbaI. An aliquot of DNA was transformedinto E. coli strain TG2 and plated on ampicillin-containing plates. Aclone that showed the proper deletion of DNA between the desiredconnection of the DRα and DAF sequences was isolated. This clone wassequenced to confirmed the presence of the desired sequences usingstandard techniques. The coding region for the DRα-DAF protein is listedin SEQ ID NO:26; the amino acid sequence encoded by SEQ ID NO:26 islisted in SEQ ID NO:27.

[0267] The plasmid containing the correct DRα-DAF construct was cut withHindIII. The ends generated by HindIII digestion were made blunt withKlenow enzyme and unphosphorylated EcoRI linkers were ligated onto theblunt ends using standard techniques. The DNA was transformed intocompetent E. coli and clones which contained the DRα-DAF sequences as aNotI-EcoRI fragment were isolated. The DRα-DAF DNA was then subclonedinto the pSRαSD5 plasmid as a NotI-EcoRI fragment to generatepSRαSD5-DRα-DAF. The map of pSRαSD5-DRα-DAF is shown in FIG. 15.

EXAMPLE 6 Construction of pSRαSD5-DRβ1-DAF

[0268] pSRαSD5-DRβ1-DAF contains a cDNA clone encoding a chimeric mouseDRβ1-DAF gene. In this chimeric protein, the extracellular domain of theDRβB1 protein is joined to sequences derived from the DAF gene. The DAFsequences provide a glycophosphatidylinositol linkage which allows thechimeric protein to be cleaved from the surface of the cell (cellsurface expression requires the expression of the DRα chain in the samecell) by treatment of the cell with phospholipase C.

[0269] a. Isolation Of A Full-Length DRβ1 cDNA

[0270] A cDNA library was prepared from poly A⁺ mRNA isolated from IBw4cells (GM03104B, NIGMS Human Genetic Mutant Cell Repository at theCoriell Institute for Medical Research, Camden, N.J.) using standardtechniques [Sambrook et al., supra at pp. 7.26-7.29]. cDNA was generatedfrom MRNA and inserted into the cloning vector λgt10 using standardtechniques [Huynh et al., in DNA Cloning: A Practical Approach (D. M.Glover, ed.), vol. 1, IRL Press Oxford (1985), pp. 49-78]. A full-lengthDRβ1 cDNA clone was isolated from the library using a full length DRβcDNA probe which was contained within the plasmid p2918.4 [Bell et al.(1985) Proc. Natl. Acad. Sci. USA 82:3405]. The resulting full-lengthDRβ1 clone was contained on a 1.2 kb NotI/EcoRI fragment.

[0271] b. Construction Of pSRαSD5-DRβ1-DAF

[0272] An in-frame connection between the extracellular coding sequenceof DRβ and the DAF sequence was performed using site-directed in vitrodeletional mutagenesis [Kunkel et al (1987), supra] as described inExample 5c.

[0273] The full length DRβ1 cDNA (section a above) was subcloned intopDAF20 (Ex. 5a) as a NotI-EcoRi fragment to generate pDAF20-DRβ1.pDAF20-DRβ1 DNA was isolated and transformed into the E. coli strainBW313. A colony was then grown overnight in LB containing 100 μg/mlampicillin. The overnight culture was diluted and incubated with helperphage as described in Example 5c to generate single-stranded pDAF20-DRβ1DNA. The ssDNA was precipitated and resuspended in TE buffer asdescribed in Example 5c.

[0274] The bridging oligonucleotide, RQBDAF2 (SEQ ID NO:28), wasphosphorylated as described in Example 5c. To anneal the bridgingoligonucleotide to the ssDNA template, 1.1 μl of phosphorylated RADAF2and 5 μl of the ssDNA prep were mixed, heated and cooled as described inExample 5c. The reaction mixture was adjusted to give, in a final volumeof 95 μl, a concentration of 16.8 mM Tris-HCl (pH 7.5), 11.6 mM MgCl₂,7.9 mM NaCl, 10.5 mM DTT and 1.1 mM rATP. Four units of T4 DNA ligase(NEB) and 3.8 units of Sequenase (US Biochemicals) were added to thereaction, which was incubated at room temp. for 5 min and 37° C. for 1hour. The reaction was adjusted to 58 mM NaCl and heated at 65° C. for10 min. The tube was cooled to 37° C. and the DNA digested with EcoRIand XbaI. An aliquot of the digested DNA was used to transform E. colistrain TG2. The transformed cells were plated on plates containingampicillin. A clone that showed the proper deletion of DNA between thedesired connection of the DRβ1 and DAF sequences was isolated. Thepresence of the desired sequences was confirmed by DNA sequencing usingstandard techniques. The coding region for the DRβ1-DAF protein islisted in SEQ ID NO:29; the amino acid sequence encoded by SEQ ID NO:29is listed in SEQ ID NO:30.

[0275] The plasmid containing the correct DRβ1-DAF construct was cutwith HindIII. The DNA was blunted with Klenow enzyme and EcoRI linkerswere added to the blunted ends using standard techniques. The DNA wastransformed into bacteria that contained the DRβ1-DAF as a NotI-EcoRIfragment were isolated. The DRβ1-DAF DNA was subcloned into pSRαSD5 as aNotI-EcoRI fragment to generate pSRαSD5-DRβ1-DAF. The map ofpSRαSD5-DRβ1-DAF is shown in FIG. 16.

EXAMPLE 7 High-Level Expression of Recombinant IL-10 In Lymphoid Cells

[0276] High levels of IL-10 were expressed in BW5147.G.1.4 cells (a Tlymphoid cell line) by co-amplification of the following threeplasmids: 1) the expression vector pJFE 14ΔIL10 which encodes mouseIL10; 2) the selection vector pMSD5-HPRT which encodes the HPRT enzymeand 3) the amplification vector pSSD7-DHFR which encodes the mouse DHFRenzyme. The plasmids were introduced into BW5147.G.1.4 cells byelectroporation. The plasmid DNA was isolated from bacterial cells usingCsCl density gradient centrifugation.

[0277] The plasmids were prepared for electroporation as follows. First,the plasmids were linearized in the same reaction tube. 200 μg of pJFE14ΔIL10 was digested with SalI. Ten μg of pMSD5-HPRT was digested withSalI. Twenty μg of pSSD7-DHFR was digested with SalI. SalI was obtainedfrom New England BioLabs and restriction digests were performedaccording to the manufacturer's instructions. The linearized plasmidswere then precipitated with ethanol and resuspended in 0.5 ml of 1XHBS(EP) buffer [20 mM HEPES (pH 7.0); 0.75 mM Na₂HPO₄/NaH₂PO₄ (pH 7.0);137 mM NaCl; 5 mM KCl and 1 gm/l dextrose].

[0278] BW5147.G.1.4 cells were grown in RPMI 1640 medium (Gibco/BRL)containing 10% FCS (HyClone) and 50 μg/ml gentamycin (Sigma). Prior toelectroporation, the cells were washed twice in ice cold 1×HBS(EP)buffer and resuspended at 2×10⁷ cells/ml in 0.5 ml of 1X HBS(EP). Thecells were then placed in a 1 ml cuvette (Sarstedt) which contained thelinearized DNAs in 0.5 ml of 1X HBS(EP). The cuvette was placed on ice.The electroporation was performed at 225 volts using an ISCO Model 493power supply. The electroporation apparatus was constructed exactly asdescribed [Chu, G. et al., (1987) Nucl. Acids Res. 15:1311]. Theelectroporation device was set on constant voltage (225V) at the 2Xsetting (i.e., both capacitors were used). Following electroporation,the cells were allowed to recover by incubation on ice for 5 to 15minutes.

[0279] The electroporated cells were then transferred to a T75 flask(Falcon) containing 30 ml of RPMI 1640 medium containing 10% FCS and 50μg/ml gentamycin. The cells were placed in a humidified atmospherecontaining 5% CO₂ at 37° C. for 36 hours. The cells were then plated in24 well plates (Falcon, Lincoln Park, N.J.) at a density of 1×10⁴cells/well in selective medium [RPMI 1640 containing 10% FCS, 100 μMhypoxanthine (Sigma) and 2 μg/ml azaserine (Sigma)]. Each well contained0.5 ml of selective medium. One week after plating the cells in the 24well plates, 0.5 ml of fresh selective medium was added.

[0280] HPRT⁺ colonies (ie., wells containing growing cells or positivewells) were visible after approximately 10 days. At day 13 (with the dayof electroporation being day zero) 100 μl of culture supernatant wasremoved and assayed for the presence of mouse IL10 using an ELISA assayperformed as described [Mosmann et al. (1990) J. Immunol. 145:2938]. Themonoclonal antibody (mcab) SXC1 (PharMingen, San Diego, Calif.) was usedas the capture antibody and biotinylated mcab SXC2 [the mcab JESS-2A5(PharMingen) may be used in place of SXC2] was used as the detectionantibody. Briefly, 20 μl of mcab SXC1 at a concentration of 2 μg/ml inPBS was allowed to bind to the wells of flexible vinyl 96 well plates(Falcon) by incubating for 30 min to 3 hours at 37° C. Excess proteinbinding sites were then blocked by adding 200 μl/well PBS,10% FCS. After30 minutes of blocking at 37° C., the plates were washed with PBS, 0.1%Tween 20 (ICN Biochemicals, Aurora, Ohio). Samples to be tested wereadded at 50 μl/well and incubated 1 hour at 37° C. Plates were washedwith PBS, 0.1% Tween 20 and 20 μl/well of PBS,0.1% Tween 20, 1 μg/mlbiotinylated mcab SXC2 was added. The plates were incubated 30 min. at37° C. The supernatants were removed and the plates were washed withPBS, 0.1% Tween 20. A {fraction (1/5000)} dilution ofstreptavidin-horseradish peroxidase conjugate (Jackson ImmunoresearchLaboratories, West Grove, Pa.) in PBS, 0.1% Tween 20, 0.1% BSA was addedat 50 μl/well and incubated 30 min. at 37° C. The plates were thenexhaustively washed with PBS, 0.1% Tween 20 and 100 μl/well of 44 mMNaH₂PO₄, 28 mM Citric Acid, 0.003% H₂O₂, 1 mg/ml 2,2′azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) was added. Theoptical densities (ODs) of the wells were measured after 1 hour using aVMAX microplate reader (Molecular Devices, Menlo Park, Calif.) with atest wavelength of 405 nm and a reference of 490 nm.

[0281] The cells from wells containing 1 to 3 apparent clones and whichcontained greater than or equal to 100 units IL10/ml were cloned bylimiting dilution using standard techniques [Cloning by LimitingDilution, in Current Protocols in Immunology (J. E. Coligan et al.,eds.) John Wiley & Sons, New York, section 2.5.10]. For the limitdilution cloning, the cells were plated at 2 cells or 4 cells per wellin a 96 well plate (Falcon) in selective medium; one 96 well plate wasset up for each cell density (2 or 4 cells/well). In total, 16independent colonies were cloned by limit dilution.

[0282] Eight days after limit dilution cloning was initiated, isolatedcolonies were picked from each of the limit dilution plates; thesecolonies were transferred to a 96 well plate; each well contained 5 mlRPMI 1640 containing 10% dialyzed FCS (HyClone) and 100 μM hypoxanthine.The use of dialyzed serum at this point increases the speed andfrequency of amplification of the transfectants; hypoxanthine is addedto the medium at this point as it is required for the growth of thecells for a few passages until the azaserine level is diluted to anegligible concentration.

[0283] Two days later, 100 μl of culture supernatant was tested for thepresence of IL-10 using an ELISA as described above. The twobest-producing clones from each of the original wells (e.g., the 24 wellplate) were chosen for further manipulation. In total 19 clones (termedselectants as these clones have survived growth in selective medium buthave not yet been subjected to amplification by growth in the presenceof methotrexate) were chosen.

[0284] Five days after the transfer of the isolated colonies (cloned bylimit dilution) to 96 well plates, the colonies were transferred to 24well plates and allowed to expand. The expanded colonies were thentransferred to 5 ml flasks (Falcon) containing 5 ml of RPMI 1640 mediumcontaining 10% dialyzed FCS. The clones produced between 100 and 200units/ml of IL-10.

[0285] The selected clones were then subjected to amplification bygrowing the cells in the presence of methotrexate. The 19 clones wereeach tested for their sensitivity to methotrexate (MTX). Five×10⁴ cellsfrom each clone was placed into a well in a series of 24 well plates.The clones were grown in the presence of RPMI 1640 medium containing 10%dialyzed FCS and either 3, 10, 30, 60 or 90 nM MTX. Six clones were ableto grow in the presence of greater than or equal to 30 nM MTX; theseclones were retained.

[0286] The six clones resistant to ≧30 nM MTX were plated in T25 flasks(Falcon) containing 5 ml of RPMI 1640 medium containing 10% dialyzed FCSand either 90, 150 or 210 nM MTX. Three flasks were set up for eachclone. The clones were allowed to grow for 15 days at these threeconcentrations of MTX and then supernatants were taken from each flaskand assayed for IL-10 production using an ELISA as above. All clonesfrom flasks containing 90 or 150 nM MTX produced between 800 and 1200units/ml of IL10. The best producing clone from each of the six originalMTX clones was selected (one from a 90 nM MTX flask and the rest from150 nM MTX flasks). These clones were then expanded to 5 mls in mediumcontaining the appropriate concentration of MTX (over a 6 day period).The clones were then transferred into medium containing either 450, 750or 1050 nM MTX. Sixteen days later supernatants from clones growing inthe presence of 1050 nm MTX were assayed for IL-10 production. Theclones were found to produce between 12,000 and 76,000 units/ml of IL-10(one clone produced 12,000 u/ml, one clone produced 15,000 u/ml andeight clones produced between 50,000 and 76,000 u/ml).

[0287] The two clones producing the highest levels of IL-10 were chosen;these clones were designated as 9-2 and 11-2. Clones 9-2 and 11-2 werethen grown in the presence of 5 μM MTX for 3 weeks, expanded and thenfrozen. Cultures were frozen as follows. Thirty milliliters of mediacontaining cells at a density of 6 to 10×10⁵ cells per ml were pelletedin a 50 ml conical tube (Falcon) at 500×g for 5 minutes. The supernatantwas poured off and the cells were resuspended in 7.5 ml of FreezingMedia (40% FCS, 53% RPMI 1640, 7% DMSO) and placed in 5 freezing vials(Nunc, Naperville, Ill.). The cells were placed in a −70° C. freezer for24 to 96 hours and then transferred to liquid nitrogen for long termstorage.

[0288] Aliquots of each clone were thawed after approximately 2 months,re-tested for IL-10 production and grown continuously in the presence of5 μM MTX. These two clones (9-2 and 11-2) continue to produce between64,000 to 86,000 units/ml of IL10.

[0289] The levels of expression of IL10 were roughly equivalent when thecells were grown at 1 or 5 μM MTX (compare 76,000 at 1 μM to 64-86,000at 5 μM). The use of concentrations of MTX greater than 5 μM appeared tomake the cells grow more slowly so that the total yield of protein wasno greater than that obtained by growing the cells in the presence of 1to 5 μM MTX.

[0290] It should be noted that selective pressure to maintain theexpression of the HPRT protein (i.e., growth in the presence of mediumcontaining hypoxanthine and azaserine) was not used after the cells weretransferred into medium containing MTX with no loss of IL-10 expression.Furthermore, because the level of IL-10 continued to rise withincreasing concentrations of MTX, the endogenous DHFR gene is not likelyto be amplified in the MTX^(r) cells. In other words, the increase inMTX-resistance is due to the amplification of the exogenous DHFR genepresent on the amplification vector pSSD7-DHFR.

EXAMPLE 8 High-Level Expression of DR Class II MHC in Lymphoid Cells

[0291] High levels of DR class II MHC molecules were expressed on thesurface of BW5147.G.1.4 cells by co-amplification of the following fourplasmids: 1) the expression vector pSRαSD5-DRα-DAF which encodes thealpha chain of the human DR molecule linked to a DAF tail; 2) theexpression vector pSRαaSD5-DRβ1-DAF which encodes the beta chain of thehuman DR molecule linked to a DAF tail; 3) the selection vectorpMSD5-HPRT which encodes the HPRT enzyme and 3) the amplification vectorpSSD7-DHFR which encodes the mouse DHFR enzyme. The plasmids wereintroduced into BW5147.G.1.4 cells by electroporation. The plasmid DNAswere isolated from bacterial cells using the standard technique of CsCldensity gradient centrifugation.

[0292] The isolated plasmid DNAs were prepared for electroporation asfollows. First the plasmids were linearized in the same reaction tube.All four plasmids were linearized with SalI. The following amounts ofplasmid were used: 200 μg of pSRαSD5-DRα-DAF; 200 μg ofpSRαSD5-DRβ1-DAF; 10 μg of pMSD5-HPRT and 25 μg of pSSD7-DHFR. Thelinearized plasmids were then precipitated with ethanol and resuspendedin 0.5 ml of 1×HBS(EP) buffer.

[0293] BW5147.G.1.4 cells were grown in RPMI-1640 medium containing 10%FCS and 50 μg/ml gentamicin. Prior to electroporation the cells werewashed twice in ice cold 1X HBS(EP) buffer and resuspended at a densityof 2×10⁷ cells/ml in 0.5 ml of 1X HBS(EP). The cells were then placed ina 1 ml cuvette (Sarstedt) which contained the linearized DNAs in 0.5 mlof 1X HBS(EP). The cuvette was placed on ice. The electroporation wasperformed as described above.

[0294] After electroporation the cells were allowed to recover byincubation on ice and then they were placed in a T75 flask (Falcon)containing 30 ml of RPMI-1640 medium containing 10% FCS and 50 μg/mlgentamicin. The cells were placed in a humidified atmosphere containing5% CO₂ at 37° C. and grown in bulk culture for 36 hours. The cells werethen plated into four 48 well plates (Costar) at a density of 10⁴cells/well in 0.5 ml selective medium [RPMI 1640 containing 10% FCS, 100μM hypoxanthine (Sigma) and 2 μg/ml azaserine (Sigma)]. The use of acell density of 1×10⁴ ensures that any colonies which arise are derivedfrom a single cell; that is this density provides for limit dilutioncloning. Any remaining cells were plated at a density of 1×10⁵cells/well in 0.5 ml of selective medium. One week after plating in the48 well plates an additional 0.5 ml of selective medium was added.

[0295] Wells containing clones capable of growth in the selective medium(selectants) were visible after 8 days. Positive colonies (i.e.,positive for growth in selective medium) were picked into 12 well plates(Costar) containing 4 ml of RPMI 1640 containing 10% dialyzed FCS(HyClone) and 100 μM hypoxanthine 10-12 days after the application ofselective medium. The use of dialyzed serum at this point increases thespeed and frequency of amplification of the selectants; hypoxanthine isadded to the medium at this point as it is required for the growth ofthe cells for a few passages until the azaserine level is diluted to anegligible concentration. The cells were allowed to grow for 3-4 days inthe 12 well plates.

[0296] Colonies which grew in the presence of hypoxanthine and azaserine(selectants) were checked for the ability to express the DR molecule onthe surface of the cell by staining cells with the monoclonal antibodyL243. L243 binds specifically to the human HLA-DR antigens [Lampson andLevy, J. Immunol., 125:293 (1980)].

[0297] The antibody was prepared as follows. Hybridoma L243 was grownand the culture supernatant collected using standard techniques [Harlowand Lane, eds., Antibodies: A Laboratory Manual, Cold Spring HarborPress, New York (1988), pp. 272, 276]. The monoclonal antibodies werepurified from the hybridoma supernatants. L243 was purified on a ProteinA-Sepharose column (Pharmacia) using the protocol supplied by themanufacturer. The purified monoclonal antibody was then biotinylatedusing standard techniques [Antibodies: A Laboratory Manual, supra at p.341]. Biotin was obtained from Vector. Biotinylated L243 was used at adilution of 1:200.

[0298] The cells were stained as follows. The contents of the wells onthe 12 well plates were gently mixed by pipeting the medium. One to 2 mlof the cell suspension was removed; this sample size contains 1-3×10⁶cells. The cells were pelleted by centrifugation at 1000 rpm for 4minutes at 4° C. One hundred μl of L243 diluted into staining media (10mM HEPES, pH 7.0, 5% calf serum, 4 mM sodium azide in Hanks balancedsalt solution) was added. The cells were incubated for 20 minutes onice. The cells were then washed by adding 1 ml of staining media andthen the cells were underlaid with 1 ml of calf serum. The cells werepelleted through the serum by centrifugation at 1000 rpm for 4 minutesat 4° C. The supernatant was removed by aspiration. The cells were thensuspended in 100 μl of fluorescein isothiocyanate (FITC) conjugatedavidin (Vector, used at 1:50 dilution). The cells were incubated for 20minutes on ice. The cells were then washed as described above.

[0299] The supernatant was removed and the cells were suspended in 200μl of staining media containing 2 μg/ml propidium iodide. Propidiumiodide is excluded from living cells but taken up by dead or dyingcells. The addition of propidium iodide allows the exclusion of deadcells (propidium iodide-bright cells) from the analysis. The cells werefiltered through nylon screen (Nitex nylon monofilament, 48 micron mesh,Fairmont Fabrics, Hercules, Calif.) prior to analysis on a FACScan™(Becton-Dickinson). An aliquot of parental BW5147.G.1.4 cells (i.e., nottransfected) was stained as above to provide a negative control.

[0300]FIG. 17 shows the results of staining a representative selectantclone, clone 5, with L243. FIG. 17 is a histogram showing the log offluorescein (x axis) plotted against the relative number of cells in thesample. Cells which express the DR molecule on the surface of theBW5147.G.1.4 cell appear as fluorescein bright cells due to staining ofthe cell surface with biotylinated-L243 followed by FITC-avidin. Asshown in FIG. 17, all of the cells in clone 5 express the transfected DRmolecule. The fact that surface expression of the DR molecule is seenshows that both the α and the β chain DR constructs are expressed insideclone 5.

[0301] Eight selectant clones having the highest levels of expression ofDR were chosen for further manipulation. These eight selectant cloneswere then tested for their sensitivity to MTX. Each clone was plated ata density of 2×10⁴ cells/well in a 24 well plate. Each well contained 1ml of medium containing RPMI-1640, 10% dialyzed FCS and MTX. The cloneswere grown in the presence of either 3, 10, 30, 60 or 90 nM MTX.Non-transfected BW5147.G.1.4 cells were also grown in the above range ofMTX as a control. Clones which grew in MTX levels at least 2-3 foldhigher than that tolerated by the parental BW5147.G.1.4 (typically lessthan or equal to 10 nM MTX) were selected for further analysis. Four ofthe selectant clones grew in greater than or equal to 30 nM MTX and wereretained; these clones are the primary transfectants chosen foramplification. All 4 clones which grew in > 30 nM MTX were analyzed forthe ability to express DR molecules on the surface by an ELISA. The cellsurface ELISA was performed as follows.

[0302] Between 5 and 20×10⁴ cells/well were put into a U-bottom 96 wellplate. The cells were pelleted in a centrifuge using a plate carrier at1000 rpm for 3 min at 4° C. The supernatant was flicked from the wells,the cells dispersed from their pellets by tapping and the plate wasplaced on ice. Fifty microliters of a {fraction (1/200)} dilution ofbiotinylated mcab L243 (Becton-Dickinson) in staining media [Hank'sBasic Salt Solution (Irvine Scientific), 10 mM HEPES, pH 7, 5% calfserum] was added to each well. The cells were incubated with thebiotinylated mcab for 20 min on ice. Ice cold staining media was addedto a final volume of 200 μl/well. The cells were pelleted and thesupernatant flicked out and the pellets dispersed as described above.The cells were washed twice more with 200 μl/well of ice cold stainingmedia. Fifty microliters of a {fraction (1/1000)} dilution ofHorseradish peroxidase conjugated Avidin (Vector Laboratories,Burlingame, Calif.) was added per well and incubated on ice for 20 min.Ice cold staining media was added to a final volume of 200 μl/well. Thecells were pelleted and the supernatant flicked out and the pelletsdispersed as described above. The cells were washed three more with 200μl/well of ice cold staining media. After the final wash, the plate wasagain tapped to disperse the cell pellets and each well received 200 μlof freshly made OPD Substrate Solution [16 mM Citric Acid, 34 mM SodiumCitrate, 0.01% H₂O, 1 mg/ml O-phenylene diamine dihydrochloride(Sigma)]. The plate was allowed to sit at room temp for 10 to 20 min.The cells were then pelleted at 1000 rpm for 3 min at 4° C. One hundredmicroliters of supernatant from each well was transferred to a fresh,flat bottom 96 well plate (Costar) and the plate was read on a VMAXmicroplate reader (Molecular Devices, Menlo Park, Calif.) at awavelength of 450 nm.

[0303] All four clones expressed the DR molecule as judged by ELISAanalysis. Each of these four clones was grown in the highest MTX levelat which obvious growth still occurred as determined by the test for MTXsensitivity above; the levels ranged from 30 to 80 nM MTX. The cloneswere then again checked for the ability to express DR on the cellsurface by staining with L243 and FACS analysis as above. One out fourfirst round amplificants, clone 5, showed both an increased resistanceto MTX and the best corresponding increase in DR expression (all fourclones showed increased DR expression). The histogram of cells fromclone 5 grown in 80 nM MTX is shown in FIG. 18. In FIG. 18 the log offluorescein (x axis) is plotted against the relative number of cells inthe sample. Growth in 80 nM MTX represents the first round ofamplification for clone 5.

[0304] The three clones which grew in higher levels of MTX but which didnot show a high coincidental increase in the expression of DR werediscarded. Clone 5 was retained and subjected to further rounds ofamplification by grow in increasing concentrations of MTX. FIGS. 19 and20 show histograms of cells from clone 5 grown in 320 nM and 1 μM MTX,respectively. The cells were stained with L243 and analyzed on a FACScanas described above. As is shown in FIGS. 19 and 20, clone 5 continued toshow a coincidental increase in DR expression and increasedMTX-resistance. Integration of the area under the peaks of fluorescencefrom each of FIGS. 17-20 showed that clone 5 achieved a 30-fold increasein DR expression between the initial selectant stage and the third roundof amplification (1 μM MTX^(r)).

[0305] Continued analysis of clone 5 demonstrated that it is extremelystable. Clone 5 grown in 1 μM MTX (referred to as the 1 μM MTXamplificant of clone 5) can be grown for 2 to 3 weeks in medium lackingMTX without any apparent drop in expression of DR (as judged by cellsurface ELISA assays).

EXAMPLE 9 Production of Large Quantities of Soluble Cell Receptor andClass II MHC Molecules

[0306] Tumors of B and T cells (i.e., lymphomas and leukemias) are oftenclonal in nature and therefore the Ig or TCR carried on the surface ofthe tumor cell can serve as a tumor-specific antigen. Soluble forms ofthe tumor-specific Ig have been used to immunize patients in order toinvoke an immune response against the tumor cell [Kwak et al. (1992) N.Engl. J. Med. 327:1209 and Hsu et aL (1996) Nature Med. 2:52]. Thetherapeutic use of soluble forms of a patient's tumor-specific antigenrequires that large quantities of the soluble antigen be produced in ashort period of time so that immunization of the patient can be carriedout quickly (i.e., before the patient's disease progress to a point thattherapy is pointless). Large quantities of soluble class II MHCmolecules are required to allow treatment of autoimmune disease usingsoluble class II molecules complexed with specific peptides [Sharma, etal. supra].

[0307] The methods of the present invention allow the production oflarge quantities of soluble forms of class II MHC molecules and TCR tobe produced in a rapid manner. These methods allow for the production ofcustomized tumor cell vaccines comprising soluble TCR for the treatmentof lymphoma and leukemia patients as well as the production of solubleclass II MHC molecules for the treatment of autoimmune disease. DNAsequences encoding the chains comprising the extracellular domains ofthe TCR or class II MHC molecules expressed by the patient's tumor cellsare cloned using the PCR. These sequences are joined to sequencesencoding a thrombin cleavage site followed by the transmembrane andcytoplasmic domains of either the α or β chain of a mammalian class IIMHC heterodimer. The sequences encoding each chain of the chimeric TCRor class II MHC molecules (i.e., the genes of interest) are insertedinto any of the SD7 vectors described herein (e.g., pSRαSD7; Ex. 1) andthe resulting vectors are co-transfected into BW5147.G.1.4 cells alongwith an amplification vector (e.g., pSSD7-DHFR, Ex. 3) and, if sodesired, a selection vector (e.g., pMSD5-HPRT; Ex. 2). The transfectedcells will express the chimeric TCR or class II MHC molecules on thecell surface. The transfected cells are subjected to selection and/oramplification in order to produce amplified cell lines which expresslarge quantities of the chimeric TCR or class II MHC molecules on thecell surface. These chimeric proteins can be cleaved from the cellsurface to produce soluble TCR or class II MHC molecules by digestionwith thrombin.

[0308] The following discussion illustrates the production of solubleTCR or class II MHC proteins using amplified cell lines. An analogousapproach can be used to produce soluble forms of any multi-chain cellsurface protein.

[0309] a. Construction Of Vectors Encoding Chimeric TCR Chains

[0310] Sequences encoding chimeric α chain of a TCR are constructedwhich comprise (from the amino- to carboxyl-termini) the extracellulardomains of the α chain of a TCR followed by 21 amino acids derived fromthe thrombin receptor which comprise a thrombin cleavage site followedby 41 amino acids comprising the transmembrane and cytoplasmic domainsof the class II MHC molecule DRα. An analogous construct is used toconstruct a chimeric β chain of a TCR comprising (from the amino- tocarboxyl-termini) the extracellular domains of the β chain of a TCRfollowed by 21 amino acids derived from the thrombin receptor whichcomprise a thrombin cleavage site followed by 42 amino acids comprisingthe transmembrane and cytoplasmic domains of the class II MIC moleculeDRβ1. Any mammalian class II MHC αβ pair can be used to providesequences encoding the transmembrane and cytoplasmic domains of the MHCmolecule which permit the association of the chimeric TCR chains. While,the number of amino acid residues comprising the transmembrane andcytoplasmic domains of the α and β chains of the class II MHC moleculesdiffers by one, both MHC junctions are at the third amino acid residuefrom the beginning of the transmembrane domain. This arrangementpreserves the glutamate residue from the α chain and the lysine from theβ chain which have been shown to have a positive effect upon heterodimerformation of class II MHC molecules [Cosson and Bonifacino (1992)Science 258:659].

[0311] A vector containing sequences encoding the thrombin and class IIMHC sequences is constructed by synthesizing the DNA sequences listed inSEQ ID NO:31 and SEQ ID NO:33. The amino acid sequence encoded by SEQ IDNO:31 is listed in SEQ ID NO:32 and amino acid sequence encoded by SEQID NO:33 is listed in SEQ ID NO:34.

[0312] SEQ ID NO:31 encodes the thrombin site-DRα chimeric sequence andSEQ ID NO:33 encodes the thrombin site-DRβ1 chimeric sequence.Inspection of these sequences shows that the sequences at the 5′ endwhich encodes the thrombin site contains the recognition site for thefollowing restriction enzymes: BamHI, PvuI and FspI. A NotI site islocated at the 3′ end of the thrombin site-DRβ₁ chimeric sequences. Thesynthetic DNA is inserted into any suitable vector (e.g., pUC 18 or pUC19) as a BamHI-NotI fragment. The thrombin site encoded by thesesequences is very efficiently cleaved by thrombin due to the presence ofthe hirudin-like domain following the thrombin cleavage site [Vu et al.(1991) Cell 64:1057 and Vu et al. (1991) Nature 353:674].

[0313] DNA sequences encoding TCR chains are isolated fromdouble-stranded cDNA generated from a cell line or a patient's tumor(double-stranded cDNA may be generated using the protocol set forth inExample 3; oligo d(T) may be used to prime first strand cDNA synthesisin place of the SBNSSdT primer). The double stranded cDNA is then usedin PCRs which contain primer pairs designed to amplify either the αchain or the β chain of the human TCR The PCR is conducted using 1unit/100 μl reaction Pfu polymerase (Stratagene) in the reaction bufferprovided by Stratagene, 5 ng/100 μl of a cloned template or 25 ng/100 μlof ds-cDNA derived from polyA+ RNA isolated from a cell line or tumor,0.1 mM of each of the four dNTPs and 0.5 μM of each primer. The PCR iscycled at 94° C. for 15 sec followed by 60° C. for 30 sec followed by75° C. for 2 min for 21 cycles.

[0314] The 5′ primer used to amplify TCR sequences contains thefollowing restriction sites at the 5′ end of the primer: XbaI, EcoRI andMluI followed 18-21 nucleotides comprising a consensus sequence derivedfrom the V regions of human TCRs. Therefore the 5′ primer will comprisesets of degenerate primers having the following sequence:5′-TCTAGAATTCACGCGT(N)₁₈₋₂₁-3′, where N is any nucleotide and the 18-21nucleotide stretch represents a consensus V region sequence. Thefollowing 3′ primer is used in conjunction with the above-describedconsensus 5′ primer to amplify the extracellular domains of human TCR αchains: 5!-CGATCGTGGATCCAAGTTTAGGTTCGTATCTGTTTCAAA-3′ (SEQ ID NO:35).The 3′ connection for the TCR α chain is made after the asparagine whichappears at position 110 of the constant (C) region of the a chain. Thefollowing 3′ primer is used in conjunction with the above-describedconsensus 5′ primer to amplify the extracellular domains of human TCR βchains: 5′-CGATCGAGGATCC AAGATGGTGGCAGACAGGACC-3′ (SEQ ID NO:36). The 3′connection for the TCR α chain is made after the isoleucine whichappears at position 147 of the C region of the β chain. These 3′ primersare designed such that in both cases (i.e., for both the α and the βchain of the TCR) the connection between the extracellular domains ofthe TCR with the thrombin site is made at the fourth amino acid residuefrom the apparent beginning of the respective transmembrane regions ofthe TCR chains. Both 3′ primers contain recognition sites for PvuI andBamHI at their 5′ ends. The restriction sites located at the 5′ ends ofthe primers allows the resulting PCR products comprising a TCR chain tobe removed as a XbaI or EcoRI or MluI (5′ end)-BamHI or PvuI (3′ end)fragment and joined with the appropriate thrombin-transmembrane DNAsequence [as a BamHI or PvuI (5′ end)-NotI (3′ end) fragment] andinserted into any of the SD7 vectors (e.g., pSRαSD7). The resultingexpression vectors (one for each of the α chains and the β chains of thechimeric TCR) are co-transfected using electroporation into BW5147.G.1.4cells along with the amplification vector pSSD7-DHFR (Ex. 3) and theselection vector pMSD5-HPRT (Ex. 2). The amount of each plasmid DNA tobe used (the plasmids are linearized before electroporation), theconditions for electroporation, selection and amplification aredescribed above. The resulting amplified cell lines will express thechimeric TCR heterodimer on the surface of the cell. The TCR issolubilized by digestion of the cells with thrombin. The thrombinsolubilized extracellular domains will have 3 (TCR β) or 4 (TCR α) novelamino acids at the C-termini.

[0315] b. Construction Of Vectors Encoding Chimeric Class H MHC Chains

[0316] Sequences encoding a chimeric a chain of a class II MHC proteinare constructed which comprise (from the amino- to carboxyl-termini) theextracellular domains of the a chain of DRα followed by 21 amino acidsderived from the thrombin receptor which comprise a thrombin cleavagesite followed by 41 amino acids comprising the transmembrane andcytoplasmic domains of the class II MHC molecule DRα. An analogousconstruct is used to construct a chimeric β chain of a class II MHCprotein comprising (from the amino- to carboxyl-termini) theextracellular domains of the β chain of DRβ₁ followed by 21 amino acidsderived from the thrombin receptor which comprise a thrombin cleavagesite followed by 42 amino acids comprising the transmembrane andcytoplasmic domains of the class II MHC molecule DRβ₁.

[0317] Sequences encoding the extracellular domains of the α and βchains of a class II MHC heterodimer are isolated using the PCR asdescribed above with the exception that the following primer pairs areused in the PCR. Sequences encoding the extracellular domain of DRα areamplified using 5′-ACGCGTCCACCATGGCC ATAAGTGGAGTCCCT-3′ (SEQ ID NO:37)(this primer contains a MluI site at the 5′ end) and5′-GGATCCAACTCTGTAGTCTCTGGGAGAG-3′ (SEQ ID NO:38) (this primer containsa BamHI site at the 5′ end). The use of these primers allows theconnection of the extracellular domain of DRα with the thrombinsite-transmembrane sequences (described above) after amino acid 191, aglutamate residue in the mature (i.e., after the removal of the signalsequence) DRα protein.

[0318] Sequences encoding the extracellular domain of DRβ₁ are amplifiedusing: 5′-ACGCGTCCACCATGGTGTGTCTGAAGCTCCTG-3′ (SEQ ID NO:39) (thisprimer contains a MluI site at the 5′ end) and 5′-GGATCCAACTTGCTCTGTGCAGATTCAGA-3′ (SEQ ID NO:40) (this primer contains a BamHI site at the 5′end). The use of these primers allows the connection of theextracellular domain of DRβ with the thrombin site-transmembranesequences (described above) after amino acid 198, a lysine residue, inthe mature DRβ protein.

[0319] The restriction sites located at the 5′ ends of the primersallows the resulting PCR products comprising the class II MHC chains tobe removed as a MluI (5′ end)-BamHII (3′ end) fragment and joined withthe appropriate thrombin-transmembrane DNA sequence [as a BamHI (5′end)-NotI (3′ end) fragment] and inserted into any of the SD7 vectors(e.g, pSRαSD7). The resulting expression vectors (one for each of the αchains and the β chains of the chimeric class II MHC protein) areco-transfected using electroporation into BW5147.G.1.4 cells along withthe amplification vector pSSD7-DHFR (Ex. 3) and the selection vectorpMSD5-HPRT (Ex. 2). The amount of each plasmid DNA to be used (theplasmids are linearized before electroporation), the conditions forelectroporation, selection and amplification are described above. Theresulting amplified cell lines will express the chimeric class IIheterodimer on the surface of the cell. The class II MHC heterodimer issolubilized by digestion of the cells with thrombin.

EXAMPLE 10 Production of Custom Multivalent Vaccines For The Treatmentof Lymphoma and Leukemia

[0320] The existing approach toward vaccination (i.e., activeimnunotherapy) of B-cell lymphoma and leukemia involves the productionof a custom vaccine comprising autologous immunoglobulin idiotype whichcorresponds to the most abundant antibody molecule expressed on thesurface of the B-cell tumor. An analogous approach for the treatment ofT-cell lymphomas and leukemias would involve the production of a customvaccine comprising autologous T cell receptor (TCR) idiotype whichcorresponds to the most abundant TCR molecule expressed on the surfaceof the B-cell tumor.

[0321] Existing methods for the production of custom vaccines for thetreatment of B-cell lymphoma employ the “rescue fusion” technique. Therescue fusion technique involves the removal of lymphoma cells bysurgical biopsy. The tumor cells are then fused with the heterohybridomacell line K6H6/B5 which has lost the ability to secrete endogenous Ig.Hybrid cells which secrete Ig corresponding to the immunophenotype ofthe tumor sample are expanded and the secreted Ig is purified for use asa vaccine [Kwak et al. (1992), supra]. The Ig produced by rescue fusionrepresents a single Ig derived from the patient's tumor; this Ig ispresumably the predominant Ig expressed by the tumor. Thus, vaccinesproduced by rescue fusion are monovalent and do not represent the fullcomplexity of Ig expressed by tumors which contain somatic variants.

[0322] In order to produce multivalent custom vaccines from smallnumbers of cells quickly and efficiently, the gene amplificationtechniques described in the preceding examples are employed. In thisexample, methods for the production of tumor-specific Ig derived from aB-cell lymphoma patient are provided. However, the general approachoutlined herein is applicable for the production of tumor-specificproteins generally (i.e., production of soluble TCR for treatment of Tcell tumors, production of Ig for treatment of B cell leukemias, etc.).

[0323] In this novel approach, the variable regions corresponding to thepatient's Ig (V_(H) and V_(L)) are molecularly cloned and joined to anappropriate constant region gene contained within an expression vector.Expression plasmids containing the patient's V_(H) region(s) joined toeither a Cγ3 or Cγ4 sequence and expression plasmids containing thepatient's V_(L) region(s) joined to either a Cκ or Cλ2 sequence arecotransfected (via electroporation) along with the selectable andamplifiable marker pM-HPRT-SSD9-DHFR into the desired cell line (e.g.,BW5147.G.1.4). The transfected cells are then subjected to selection andamplification as described in the preceding examples. The methodoutlined below permits the production of a multivalent vaccine whichreflects the degree of somatic variation found within the patient'stumor. These novel multivalent vaccine preparations provide superiorvaccines for the treatment of B-cell lymphoma and should reduce the rateof relapse observed when the current generation of monovalent vaccinesare employed.

[0324] a) Construction of Expression and Selection/AmplificationPlasmids

[0325] For the following constructions, unless otherwise stated, allenzymes are obtained from New England Biolabs (NEB) and used inconjunction with the buffers and reaction conditions recommended by themanufacturer.

[0326] i) Construction of pSRαSD9

[0327] Two micrograms of pSRαSD7 (Ex. X) is cut with SalI and HindIII(NEB enzymes, buffers & conditions). The plasmid is spermineprecipitated (Ex. 5) and resuspended in 34 μl H₂O and 4 μl of 10× T4 DNAligase buffer. Equal molar amounts (6.3 ng each) of the unphosphorylatedoligonucleotides SXAPH5 (SEQ ID NO:42) and SXAPH3 (SEQ ID NO:43) areadded. The reaction is chilled on ice, 400 units of T4 DNA ligase isadded and the tube is placed at 14° C. overnight. The ligation istransformed into bacteria and clones screened for the presence of theadded AscI & PacI restriction sites. The resulting plasmid is calledpSRαSD9. FIG. 21 provides a schematic map of pSRαSD9.

[0328] ii) Construction of pSRαSD9CG3C, pSRαSD9CG4C, pSRαSD9CKC andpSRαSD9CL2C

[0329] The plasmids pSRαSD9CG3C, pSRαSD9CG4C, pSRαSD9CKC and pSRαSD9CL2Ccontain sequences encoding the Cγ3, Cγ4, Cκ or Cλ2 constant regions,respectively. The constant regions contained within these expressionvectors are encoded by synthetic DNA sequences which encode the sameamino acid sequences as that found in the native proteins; however, theDNA sequences have been modified to utilize codons which are found mostfrequently in highly expressed manunalian proteins [Haas et al. (1996)Curr. Biol. 6:315 and Zolotukhin et al. (1996) J. Virol. 70:4646]. TheDNA sequence encoding the Cγ3 region is listed in SEQ ID NO:44; theamino acid sequence encoded by SEQ ID NO:44 is listed in SEQ ID NO:45.The DNA sequence encoding the Cγ4 region is listed in SEQ ID NO:46; theamino acid sequence encoded by SEQ ID NO:46 is listed in SEQ ID NO:47.The DNA sequence encoding the Cκ region is listed in SEQ ID NO:48; theamino acid sequence encoded by SEQ ID NO:48 is listed in SEQ ID NO:49.The DNA sequence encoding the Cλ2 region is listed in SEQ ID NO:50; theamino acid sequence encoded by SEQ ID NO:50 is listed in SEQ ID NO:51.

[0330] Double stranded DNA corresponding to SEQ ID NOS:44, 46, 48 and 50are synthesized (Operon Technologies). Each synthetic DNA sequence iscut with NotI and BglII, run through a 0.8% SeaPlacque Agarose gel (FMC)and recovered using β-agarase as described below. Each C region sequenceis ligated to the two DNA restriction fragments generated from pSRαSD9as follows. A 2 μg aliquot of pSRαSD9 is cut with HindIII and BamHI anda 2314 bp band is isolated. A second 2 μg aliquot of pSRαSD9 is cut withHindIII and NotI and an 854 bp band is isolated. These fragments areisolated by running each digest on a 0.8% SeaPlacque Agarose (FMC), theappropriate bands are cut out and combined in a microfuge tube. Theagarose is remove by β-Agarase (NEB) digestion and the DNA is recoveredby isopropanol precipitation exactly as indicated by NEB.

[0331] The ligation of SEQ ID NO:44 (digested with NotI and BglII) withthe above fragments of pSRαSD9 generates pSRαSD9CG3C (map shown in FIG.22). The ligation of SEQ ID NO:45 (digested with NotI and BglII) withthe above fragments of pSRαSD9 generates pSRαSD9CG4C (map shown in FIG.23). The ligation of SEQ ID NO:46 (digested with NotI and BglII) withthe above fragments of pSRαSD9 generates pSRαSD9CKC (map shown in FIG.24). The ligation of SEQ ID NO:47 (digested with NotI and BglII) withthe above fragments of pSRαSD9 generates pSRαSD9CL2C (map shown in FIG.25).

[0332] iii) Construction of pM-HPRT-SSD9-DHFR

[0333] pM-HPRT-SSD9-DHFR contains the hprt gene under the control of theMoloney enhancer/promoter and the dhfr gene under the control of theSV40 enhancer/promoter. pM-HPRT-SSD9-DHFR is constructed by firstsubcloning the HPRT cDNA (Ex. 2) into pMSD8 (described below) to createpMSD8-HPRT. The small DNA fragment located between the SalI and HindIIIsites on pMSD8-HPRT is then replaced with a sequence containing AscI andPacI sites as follows. pMSD8-HPRT is digested with SalI and HindIII andthe SXAPH5 and SAXPH3 oligonucleotides (SEQ ID NOS:42 and 43) areligated to the ends of the digested pMSD8-HPRT (as described in sectioni above) to create pMSD9-HPRT. The ˜2450 bp SalI-ClaI fragmentcontaining the AscI and PacI sites, the Moloney enhancer/promoter, theHPRT cDNA and the EF1α poly A region is inserted between the SalI andClaI sites of pSSD7-DHFR (Ex.) to generate pM-HPRT-SSD9-DHFR. FIG. 26provides a map of pM-HPRT-SSD9-DHFR.

[0334] pMSD8 is similar to pMSD5 but contains the poly A site from thehuman elongation factor 1α gene. pMSD8 was constructed as follows: A 292bp fragment containing the poly A site from the human elongation factor1α gene (SEQ ID NO:78) was isolated from MOU cell (GM 08605, NIGMS HumanGenetic Mutant Cell Repository, Camden, N.J.) genomic DNA using PCR. MOUgenomic DNA was isolated using conventional techniques. The PCR wasconducted using 10 μg MOU genomic DNA and 1 μM final concentration ofeach primer in a 400 μl reaction. Reaction conditions were 94° C. for 1minute, 60° C. for 1 minute, 72° C. for 1.5 minutes, 30 cycles. Taq DNApolymerase was obtained from Perkin-Elmer. The followingoligonucleotides were used to prime the PCR: 5EF1αPolyA: 5′GAATTCTTTTTTGCGTGTGGCAG 3′ (SEQ ID NO:79) and 3EF1αPolyA: 5′ATCGATATTCCTTCCCCTTCC 3′ (SEQ ID NO:80). The 3EF1αPolyA oligonucleotidegenerates a ClaI site at the 3′ end of the poly A site and the5EF1αPolyA oligonucleotide generates an EcoRI site at the 5′ end of thepoly A site. Digestion of the PCR product with EcoRI and ClaI yields a292 bp EcoRI/ClaI fragment.

[0335] pSSD5 (Ex. 1) was digested with PvuII and a ClaI linkers (NEB,unphosphorylated) were ligated to the PvuII ends to convert the PvuIIsite located at the 3′ end of the SV40 poly A site to a ClaI site. Theresulting construct was then digested with SalI and ClaI and the ˜2.1 kbfragment containing the plasmid backbone (e.g., the AmPR gene andplasmid ORI) was isolated and ligated to an ˜870 bp SalI/EcoRI fragmentcontaining the Moloney enhancer/promoter, splice donor/acceptor andpolylinker isolated from pMSD5 (Ex. 1) together with the 292 bpEcoRI/ClaI fragment containing the poly A site of the human elongationfactor 1α gene to generate pMSD8.

[0336] b) Collection of Tumor Cells

[0337] Cells are collected by either surgical biopsy of enlarged lymphnodes or by fine needle biopsy of effected lymph nodes. The biopsysample is rapidly frozen on dry ice.

[0338] c) Isolation of RNA From Tumor Cells

[0339] RNA is isolated from the biopsy sample by using a variety ofstandard techniques or commercially available kits. For example, kitswhich allow the isolation of RNA from tissue samples are available fromQiagen, Inc. (Chatsworth, Calif.) and Stratagene (LaJolla, Calif.),respectively. Total RNA may be isolated from tissues and tumors by anumber of methods known to those skilled in the art and commercial kitsare available to facilitate the isolation. For example, the RNeasy® kit(Qiagen Inc., Chatsworth, Calif.) provides protocol, reagents andplasticware to permit the isolation of total RNA from tissues, culturedcells or bacteria, with no modification to the manufacturer'sinstructions, in approximately 20 minutes. Should it be desirable tofurther enrich for messenger RNAs, the polyadenylated RNAs in themixture may be specifically isolated by binding to anoligo-deoxythymidine matrix, through the use of a kit such as theOligotex® kit (Qiagen). Comparable isolation kits for both of thesesteps are available through a number of commercial suppliers.

[0340] In addition, RNA may be extracted from samples, including biopsyspecimens, conveniently by lysing the homogenized tissue in a buffercontaining 0.22 M NaCl, 0.75 mM MgCl₂, 0.1 M Tris-HCl, pH 8.0, 12.5 mMEDTA, 0.25% NP40, 1% SDS, 0.5 mM DTT, 500 u/ml placental RNAse inhibitorand 200 μg/ml Proteinase K. Following incubation at 37° C. for 30 min,the RNA is extracted with phenol:chloroform (1:1) and the RNA isrecovered by ethanol precipitation.

[0341] A particularly preferred method for the isolation of totalcellular RNA from patient tumor samples is the RNAzol method (Teltest,Inc., Friendswood, Tex.) which is performed according to themanufacturer's instructions.

[0342] d) Cloning of Ig Genes from Tumor Cells

[0343] Because the first and third complementarity determining regions(CDRs) of rearranged immunoglobulin genes are flanked by conservedsequences, it is possible to design PCR primers capable of amplifyingcDNA for the variable regions from mRNA derived from Ig-expressing tumorcells without any specific knowledge of the nucleotide sequence of thatspecific antibody. Primers suitable for isolating the variable regionsfrom a patient's tumor are provided below.

[0344] Using total cellular RNA isolated from the tumor, double stranded(ds) cDNA is generated as described in Example 3 with the exception that20 μg of total cellular RNA is used instead of poly A⁺ RNA. Five percentof the ds cDNA preparation is used for each PCR reaction.[Alternatively, ds cDNA may be produced using the technique of RT-PCR(reverse transcription-PCR); kits which permit the user to start withtissue and produce a PCR product are available from Perkin Elmer(Norwalk, Conn.) and Stratagene (LaJolla, Calif.). The RT-PCR techniquegenerates a single-stranded cDNA corresponding to a chosen segment ofthe coding region of a gene by using reverse transcription of RNA; thesingle-stranded cDNA is then used as template in the PCR].

[0345] PCR reactions are carried out in a final volume of 50 μl andcontain 1× Pfu Buffer (Stratagene), all 4 dNTPs at 100 μM each, primersat 0.5μM each, Pfu polymerase (Stratagene) and 5% of the ds cDNApreparation. The reactions are thermocycled as follows: 94° C., 15 sec;60° C., 30 sec; 75° C., 1.5 min for 15-30 cycles. Aliquots (5 μl) areremoved after 15, 20, 25 and 30 cycles to examine the appearance of theprimary PCR product. Preparative reactions of 200 μl using the correct Vregion primers will be then run for cloning purposes.

[0346] Prior to conducting a PCR reaction to obtain Ig sequences from apatient's tumor, the tumor is immunophenotyped using commerciallyavailable antibodies to determine the heavy chain and light chainisotypes; this allows the number of PCRs to be minimized. For example,if the Ig expressed by the patient's tumor utilizes a μ heavy chain anda κ light chain, then PCR reactions described below which contain Cγ andCλ primers need not be run. However, the use of PCR primerscorresponding to heavy and light chain isotypes which are not utilized(according to the immunophenotyping results) by the patient's tumorserves as a convenient means to confirm the immunophenotyping results.

[0347] PCR primers utilized to clone variable regions of the patient'stumor-specific Ig are summarized below in Tables 1 through 3: TABLE 1Heavy Chain Primers: VH1L 5′-TCT AGA ATT CAC GCG TCC ACC ATG GAC TGG ACCSEQ ID TGG AG-3′ NO: 52 VH2L 5′-TCT AGA ATT CAC GCG TCC ACC ATG GAC ACACTT TGC SEQ ID TAC AC-3′ NO: 53 VH3L 5′-TCT AGA ATT CAC GCG TCC ACC ATGGAG TTT GGG CTG SEQ ID AGC TGG-3′ NO: 54 VH4L 5′-TCT AGA ATT CAC GCG TCCACC ATG AAA CAC CTG SEQ ID TGG TTC TTC CT-3′ NO: 55 VH5L 5′-TCT AGA ATTCAC GCG TCC ACC ATG GGG TCA ACC SEQ ID GCC ATC CT-3′ NO: 56 VH6L 5′-TCTAGA ATT CAC GCG TCC ACC ATG TCT GTC TCC TTC SEQ ID CTC ATC TT-3′ NO: 57C_(γ) 5′-GCC TGA GTT CCA CGA CAC CGT CAC-3′ SEQ ID NO: 58 C_(μ) 5′-GGGGAA AAG GGT TGG GGC GGA TGC-3′ SEQ ID NO: 59 JH1245 5′-GAG GGG CCC TTGGTC GAC GCT GAG GAG ACG GTG SEQ ID ACC AGG-3′ NO: 60 JH3 5′-GAG GGG CCCTTG GTC GAC GCT GAA GAG ACG GTG SEQ ID ACC ATT G-3′ NO: 61 JH6 5′-GAGGGG CCC TTG GTC GAC GCT GAG GAG ACG GTG SEQ ID ACC GTG-3′ NO: 62

[0348] TABLE 2 Kappa Chain Primers: VκI 5′-TCT AGA ATT CAC GCG TCC ACCATGGAC ATG AGG GTC SEQ ID CCC GCT CAG-3′ NO: 63 VκII 5′-TCT AGA ATT CACGCG TCC ACC ATG AGG CTC CCT GCT SEQ ID CAG C-3′ NO: 64 VκIII 5′-TCT AGAATT CAC GCG TCC ACC ATG GAA GCC CCA SEQ ID GCG CAG CTT-3′ NO: 65 VκIV5′-TCT AGA ATT CAC GCG TCC ACC ATG GTG TTG CAG ACC SEQ ID CAG GT-3′ NO:66 VκV 5′-TCT AGA ATT CAC GCG TCC ACC ATG GGG TCC CAG GTT SEQ ID CACCT-3′ NO: 67 VκVIa 5′-TCT AGA ATT CAC GCG TCC ACC ATG TTG CCA TCA CAASEQ ID CTC ATT G-3′ NO: 68 VκVIb 5′-TCT AGA ATT CAC GCG TCC ACC ATG GTGTCC CCGTTG SEQ ID CAA TT-3′ NO: 69 Cκ 5′-GGT TCC GGA CTT AAG CTG CTC ATCAGA TGG CGG G-3′ SEQ ID NO: 70

[0349] TABLE 3 Lambda Chain Primers: VL1 5′-TGT AGA ATT CAC GCG TCC ACCATG GCC TGCTCT CCT SEQ ID CTC CTC CT-3′ NO: 71 VL2 5′-TCT AGA ATT CACGCG TCC ACC ATG GCC TGG GCT CTG SEQ ID CTG CTC CT-3′ NO: 72 VL3 5′-TCTAGA ATT CAC GCG TCC ACC ATG GCC TGG ATC CTT SEQ ID CTC CTC CTC-3′ NO: 73VL4 5′-TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG ACC CCT SEQ ID CTC TGGCTC-3′ NO: 74 VL6 5′-TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG GCC CCA SEQID CTA CT-3′ NO: 75 VL8 5′-TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG ATGSEQ ID ATG CTT CTC CT-3′ NO: 76 Cλ 5′-GGC GCC GCC TTG GGC TGA CCT AGGACG GT-3′ SEQ ID NO: 77

[0350] The VH1-6L primers contain recognition sites for XbaI, EcoRI andMluI at their 5′ ends. The three JH primers contain recognition sitesfor ApaI and SalI at their 5′ ends. The seven Vκ primers containrecognition sites for XbaI, EcoRI and MluI at their 5′ ends. The Cκprimer contains recognition sites for BspEI and AflIII at the 5′ end.The six VL primers contain recognition sites for XbaI, EcoRI and MluI attheir 5′ ends. The Cλ primer contains recognition sites for KasI andAvrII at the 5′ end.

[0351] For each tumor sample, five V_(H) PCR reactions are run. EachV_(H) reaction will contain the Cμ and Cγ primers. The Cμ primer (SEQ IDNO:59) should result in ˜590 bp product for the heavy chain V (V_(H))region expressed in an IgM positive tumor. The Cγ primer (SEQ ID NO:58)should result in ˜480 bp product for the heavy chain V region expressedin an IgG positive tumor. The VH1, VH2, VH3, and VH4 primers (SEQ IDNOS: 52-55 , respectively) are used in separate PCR reactions and theVH5 and VH6 primers (SEQ ID NOS:56 and 57, respectively) are usedtogether in the same reaction. The V_(H) primer(s), which when used inconnection with a C_(H) region primer, gives a PCR product of theexpected size is then be used in three separate PCR reactions containingeither the JH1245, JH3 or JH6 primers (SEQ ID NOS:60-62, respectively)to generate a PCR product corresponding to the variable (V), diversity(D) and joining (J) regions present in the Ig(s) expressed by thepatient's tumor. The VDJ reaction product is then subeloned into thepSRαSD9CG3C vector or pSRαSD9CG4C vector using the 5′ XbaI, EcoRI orMluI sites and the 3′ SalI or ApaI sites to provide an expression vectorencoding the patient's heavy chain variable domain linked to either a γ3or γ4 constant domain. As is understood by those in the art, the PCRproduct is subcloned into the expression vector using restrictionenzymes which lack sites internal to the PCR product (i.e., within theIg sequences). The PCR products are digested with restriction enzymesthat have sites located within the PCR primers to confirm that the PCRproduct lacks an internal site for a given restriction enzyme prior tosubcloning the PCR product into the desired expression vector. It isanticipated that the 5′ MluI site can be employed for each PCR productgiven that MluI sites are very infrequently found in the genome; howeverthe 5′ primers also contain XbaI and EcoRI sites in the event aparticular PCR product contains an internal MluI site. The followingrestriction enzymes (which have recognition sites in the above-described3′ PCR primers) are examined first for their inability to cut internallyto the PCR products: SalI for heavy chain PCR products; AflII for kappalight chain PCR products; AvrII for lambda light chain PCR products. Asdiscussed above, each 3′ PCR primer provides alternative restrictionenzyme sites.

[0352] With regard to choosing an expression vector, the pSRαSD9CG3Cvector is initially chosen as Cγ3 is the least frequently used isotypein humans (Cγ4 is the next least frequently utilized isotype, with Cγ1and Cγ2 being the most frequently used isotypes) and therefore ELISAsperformed following immunization with a vaccine comprising Cγ3 areeasier to conduct and interpret as the patient's anti-idiotype responsewill mainly consist of the γ1 and γ2 isotypes. However, Cγ4 may bechosen over Cγ3 if a given Cγ3 construct produces an Ig protein whichtends to fall out of solution upon purification.

[0353] For each tumor sample, five Vκ PCR reactions are run. Each Vκ PCRreaction will contain the Cκ primer (SEQ ID NO:70). The VκI, VκII, andVκIII primers (SEQ ID NOS:63-65, respectively) will be run in separatereactions. The VκIV and VκV primers (SEQ ID NOS:66 and 67, respectively)are combined in one PCR reaction and the VκVIa and VκVIb primers (SEQ IDNOS:68 and 69, respectively) in another. The PCR reaction which yields aPCR product of the expected size (˜480 bp) is used as the source of DNAencoding the variable domain derived from the light chain of thepatient's Ig. The positive reaction product is subcloned into thepSRαSD9CKC vector using the 5′ XbaI, EcoRI or MluI sites and the 3′AflII or BspEI sites.

[0354] For each tumor sample, six Vλ PCR reactions are run. Each Vκ PCRreaction will contain the Cλ primer (SEQ ID NO:77). The VL1, VL2, VL3,VL4, VL6 and VL8 primers (SEQ ID NOS:71-76, respectively) are used inseparate reactions. The PCR reaction which yields a PCR product of theexpected size (˜420 bp) is used as the source of DNA encoding thevariable domain derived from the light chain of the patient's Ig. Thepositive reaction product will be subcloned into the pSRαSD9CL2C vectorusing the 5′ XbaI, EcoRI or MluI sites and the 3′ AvrII or KasI sites.It is understood by those skilled in the art that the tumor cells willexpress either a κ or a λ light chain. Therefore, it is expected that aPCR product will be recovered from either the Vκ or Vλ PCRs but not fromboth.

[0355] e) Expression and Amplification of Tumor-Specific Ig in MammalianCells

[0356] Once expression vectors containing sequences derived from thevariable regions of the heavy and light chains found in the patient'stumor are constructed, these plasmids are used to transform E. coliusing conventional techniques. Between 18 and 24 colonies from eachsubcloning are screened for heavy and light chain inserts as appropriateby restriction enzyme analysis of miniprep DNA (from 1-1.5 ml cultures).Equal aliquots of the positive subclones are used to inoculate largercultures (˜250 mls) from which the DNA for electroporation is prepared.This allows for the isolation of the somatic variants in the tumorpopulation and result in transfectants (e.g., BW5147.G.1.4transfectants) expressing these somatic variants.

[0357] To further define the presence of somatic variants, 20 μl PCRreactions are run using ˜100 pg of each miniprep DNA and the appropriateV region and C region primers. Digestion of the resulting PCR productswith several four base recognition restriction enzymes allows thedifferentiation of somatic variants. In addition, DNA sequencing can beperformed on individual subclones to demonstrate the presence of somaticvariants within the pool of subclones containing the cloned heavy andlight chain variable regions.

[0358] Plasmids encoding the chimeric heavy and light chains derivedfrom the patient's Ig are electroporated along with pM-HPRT-SSD9-DHFRinto BW5147.G.1.4 cells as follows. The Ig expression plasmids (whichcomprise a mixture of vectors containing the somatic variants foundwithin the tumor Ig) are linearized by digestion with AscI or PacI.pM-HPRT-SSD9-DHFR is linearized with AscI or PacI. pM-HPRT-SSD9-DHFR andthe Ig expression plasmids are used at a ratio of 1:20-50. Approximately15 μg of pM-HPRT-SSD9-DHFR (10-20 μg) is used while a total of ˜500 μgof the expression vectors are used. The linearized plasmids aredigested, precipitated and resuspended in 0.5 ml electroporation buffer[i.e., IX HBS(EP)] as described in Example 7. The linearized plasmids in0.5 ml electroporation buffer are mixed with 2×10⁷ cells (e.g.,BW5147.G.1.4) in 0.5 ml electroporation buffer and electroporated asdescribed in Example 7. The cells are then grown in selective mediumfollowed by growth in medium containing MTX as described in Examples 7and 8. Clones which grow in the selective medium are checked for theability to express the cloned Ig proteins using standard methods (e.g.,by ELISA). Primary selectants expressing high levels of the cloned Igproteins are then grown in medium containing MTX as described inExamples 7 and 8 to amplify the transfected genes. The presence of theselectable and amplifiable markers on a single piece of DNA (i.e.,pM-HPRT-SSD9-DHFR), obviates concerns that primary transfectants (i.e.,cells capable of growing in medium containing Hx and Az) which expressthe genes of interest (i.e., the Ig proteins) at high levels have failedto integrate a DBFR gene.

[0359] f) Purification of Tumor-Specific Ig From Amplified Cell Lines

[0360] The tumor-specific Ig expressed by the amplified cell lines(using either the pSRαSD9CG3C or pSRαSD9CG4C vectors) is purified bychromatography of culture supernatants on Protein G Sepharose(Pharmacia); Protein G binds to both IgG₃ and IgG₄. The chromatographyis conducted according to the manufacturer's instructions. When thetumor-specific Ig is produced using the pSRαSD9CG4C vector, Protein ASepharose (Pharmacia) may also be employed for purification.

[0361] g) Administration of Tumor-Specific Ig (Multivalent Vaccine)

[0362] The purified tumor immunoglobulin-idiotype protein may beconjugated to a protein carrier such as keyhole limpet hemocyanin (KLH)(Calbiochem, San Diego, Calif.) prior to administration to the patient.If the immunoglobulin-idiotype protein is to be conjugated with KLH, theKLH is depleted of endotoxin using methods known to the art [Kwak et al.(1992), supra]. For example, the KLH is applied to a QAE Zeta Prep 15disk (LKB, Broma, Sweeden) to produce a preparation of KLH containingless than 1000 endotoxin units per milliliter. Equal volumes of filtersterilized purified KLH and purified immunoglobulin-idiotype protein(each at 1 mg/ml) are mixed together. Sterile glutaraldehyde is added ata final concentration of 0.1%. The Ig-KLH conjugate is then dialyzedextensively against physiologic saline to remove excess glutaraldehyde.

[0363] Purified immunoglobulin-idiotype protein (conjugated orunconjugated) is mixed with an immunologic adjuvant such as SAF-1(Syntex adjuvant formulation 1; Roche) or other adjuvant presently orsubsequently approved for administration to humans [e.g., QS-21(Perlmmune, Inc., Rockville, Md.)]. The purified immunoglobulin-idiotypeprotein is emulsified in the desired adjuvant and injectedsubcutaneously at 0, 2, 6, 10 and 14 weeks. Booster injects may be givenat 24 and 28 weeks. Each injection contains 0.5 mg of purified,tumor-specific idiotype immunoglobulin (which may be conjugated 1:1 withKLH).

[0364] An alternative to the use of KLH as a foreign carrier protein toboost the immune response to the immunoglobulin idiotype protein is theuse of a fusion protein comprising idiotype protein and a cytokine(e.g., GM-CSF, IL-2 or IL-4) [PCT International ApplicationPCT/US93/09895, Publication No. WO 94/08601 and Tao and Levy (1993)Nature 362:755 and Chen et al. (1994) J. Immunol. 153:4775]. In thesefusion proteins, sequences encoding the desired cytokine are added tothe 3′ end of sequences encoding the immunoglobulin idiotype protein.The present invention contemplates the use of idiotype-cytokine fusionproteins for the treatment of B-cell lymphoma. The sequences encodingthe heavy chain of the patient's immunoglobulin protein are cloned asdescribed above and inserted into an expression vector containingsequences encoding the desired cytokine such that a fusion proteincomprising, from amino- to carboxy-terminus, the heavy chain of thepatient's tumor-specific immunoglobulin and the desired cytokine.

[0365] An alternative to the use of foreign carrier proteins, cytokines,or immunologic adjuvants is the use of autologous dendritic cells pulsedwith the purified immunoglobulin-idiotype protein [see for example, Hsuet al. (1996), supra and PCT International Application PCT/US91/01683,Publication No. WO 91/13632]. Methods for the isolation of humandendritic cells from peripheral blood are known to the art [Mehta et al.(1994) J. Immunol. 153:996 and Takamizawa et al. (1995) J. Clin. Invest.95:296]. Briefly, the patient is leukapheresed using a cell separator(COBE). Peripheral blood mononuclear cells (PBMCs) are collected byseparation through Ficoll-Hypaque (Pharmacia). Monocytes are thenremoved by centrifugation through discontinuous Percoll (Pharmacia)gradients. The monocyte-depleted PBMCs are then placed in medium (RPMI1640 containing 10% autologous patient serum) containing idiotypeprotein (2 μg/ml). Following incubation for 24 hours at 37° C. in ahumidified atmosphere containing 10% CO₂, the dendritic cells areseparated from lymphocytes by sequential centrifugation through 15% and14% (wt/vol) metrizamide gradients. The preparation is then incubatedfor 14-18 hours in medium containing 50 μg/ml idiotype protein. Thecells are then washed to remove free antigen (i.e., idiotype protein)and placed in sterile saline containing 5% autologous serum andadministered intravenously.

[0366] Each patient is followed to determine the production ofidiotype-specific antibody; the in vitro proliferative responses (toKLH, if used, and to immunoglobulin idiotype using 0 to 100 μg ofsoluble protein per milliliter in 5 day in vitro cultures) of PBMCsisolated from the treated patients may also be determined. These assaysare conducted immediately before each immunization and 1 to 2 monthsfollowing the last immunization. Patients are monitored for diseaseactivity by physical examination, routine laboratory studies and routineradiographic studies. Regression of lymph nodes or cutaneouslymphomatous masses may be confirmed by computed tomography (CT). Inaddition, residual disease may be measured using a tumor-specific CDR3analysis as described by Hsu et al. (1996), supra.

[0367] h) Treatment of T-cell Tumors

[0368] Vaccines comprising soluble T cell receptor (TCR) proteinsderived from a patient's T cell tumor (i.e., a T cell leukemia orlymphoma) are produced using the methods described in Example 9 with theexception that pM-HPRT-SSD9-DHFR is used in place of separate selectionand amplification vectors as described above. The thrombin solubilizedTCR proteins are purified by chromatography on a resin comprising amonoclonal antibody (mcab) directed against a monomorphic determinant onhuman αβ TCRs [e.g., mcab T10B9.1A-31 (Pharmingen, San Diego, Calif.);mcab BMA031 (Immunotech, Westbrook, Me.); mcabs BW242/412, 8A3 or 3A8(Serotec, Washington, DC). Antibodies directed against monomorphic(i.e., invariant) determinants on TCRs recognize all αβ TCRs.

[0369] The purified tumor-specific idiotype TCR protein is administeredas described above for the purified tumor-specific idiotype Ig protein(ie., mixing with an immunologic adjuvant, conjugation to a proteincarrier, the use of TCR-cytokine fusion proteins, the use of dendriticcells pulsed with the purified TCR protein such as SAF-1, etc.).Patients are followed to determine the production of idiotype-specificantibody as described above. Patients are monitored for disease activityby physical examination, routine laboratory studies and routineradiographic studies.

[0370] From the above, it is clear that the present invention providesimproved methods for the amplification and expression of recombinantgenes in cells. The resulting amplified cell lines provide largequantities of recombinant proteins in a short period of time. Theability to produce large quantities of recombinant proteins in a shortperiod of time is particularly advantageous when proteins unique to apatient's tumors are to be used for therapeutic purposes, such as forvaccination.

[0371] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 80 1 28 DNA Artificial Sequence Synthetic 1 tctagagcgg ccgcggaggccgaattcg 28 2 36 DNA Artificial Sequence Synthetic 2 gatccgaattcggcctccgc ggccgctcta gatgca 36 3 677 DNA SV40 Poly A 3 ggatccagacatgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 60 aaaaaaatgctttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 120 ctgcaataaacaagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 180 ggtgtgggaggttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga 240 tcatgaacagactgtgagga ctgaggggcc tgaaatgagc cttgggactg tgaatcaatg 300 cctgtttcatgccctgagtc ttccatgttc ttctccccac catcttcatt tttatcagca 360 ttttcctggctgtcttcatc atcatcatca ctgtttctta gccaatctaa aactccaatt 420 cccatagccacattaaactt cattttttga tacactgaca aactaaactc tttgtccaat 480 ctctctttccactccacaat tctgctctga atactttgag caaactcagc cacaggtctg 540 taccaaattaacataagaag caaagcaatg ccactttgaa ttattctctt ttctaacaaa 600 aactcactgcgttccaggca atgctttaaa taatctttgg gcctaaaatc tatttgtttt 660 acaaatctggcctgcag 677 4 39 DNA Artificial Sequence Synthetic 4 ctagaattcacgcgtaggcc tccgcggccg cgcgcatgc 39 5 39 DNA Artificial SequenceSynthetic 5 aattgcatgc gcgcggccgc ggaggcctac gcgtgaatt 39 6 633 DNA SRalpha promoter 6 caagcttgct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggctccccagcag 60 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtggaaagtccccag 120 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagcaaccatagtcc 180 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccattctccgcccc 240 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcctctgagctat 300 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaagctcctcgagc 360 tcgcatctct ccttcacgcg cccgccgccc tacctgaggc cgccatccacgccggttgag 420 tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact gcgtccgccgtctaggtaag 480 tttagagctc aggtcgagac cgggcctttg tccggcgctc ccttggagcctacctagact 540 cagccggctc tccacgcttt gcctgaccct gcttgctcaa ctctacgtctttgtttcgtt 600 ttctgttctg cgccgttaca gatcgcctcg agg 633 7 635 DNAMoloney LTR virus 7 caagcttgcg attagtccaa tttgttaaag acaggatatcagtggtccag gctctagttt 60 tgactcaaca atatcaccag ctgaagccta tagagtacgagccatagata aaataaaaga 120 ttttatttag tctccagaaa aaggggggaa tgaaagaccccacctgtagg tttggcaagc 180 tagcttaagt aacgccattt tgcaaggcat ggaaaaatacataactgaga atagagaagt 240 tcagatcaag gtcaggaaca gatggaacag ctgaatatgggccaaacagg atatctgtgg 300 taagcagttc ctgccccggc tcagggccaa gaacagatggaacagctgaa tatgggccaa 360 acaggatatc tgtggtaagc agttcctgcc ccggctcagggccaagaaca gatggtcccc 420 agatgcggtc cagccctcag cagtttctag agaaccatcagatgtttcca gggtgcccca 480 aggacctgaa atgaccctgt gccttatttg aactaaccaatcagttcgct tctcgcttct 540 gttcgcgcgc ttctgctccc cgagctcaat aaaagagcccacaacccctc actcggggcg 600 ccagtcctcc gattgactga gtcgccccct cgagg 635 8483 DNA Homo sapiens 8 aagctttgga gctaagccag caatggtaga gggaagattctgcacgtccc ttccaggcgg 60 cctccccgtc accacccccc ccaacccgcc ccgaccggagctgagagtaa ttcatacaaa 120 aggactcgcc cctgccttgg ggaatcccag ggaccgtcgttaaactccca ctaacgtaga 180 acccagagat cgctgcgttc ccgccccctc acccgcccgctctcgtcatc actgaggtgg 240 agaagagcat gcgtgaggct ccggtgcccg tcagtgggcagagcgcacat cgcccacagt 300 ccccgagaag ttggggggag gggtcggcaa ttgaaccggtgcctagagaa ggtggcgcgg 360 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctttttcccgagg gtgggggaga 420 accgtatata agtgcagtag tcgccgtgaa cgttctttttcgcaacgggt ttgccgcctc 480 gag 483 9 24 DNA Artificial Sequence Synthetic9 aagctttgga gctaagccag caat 24 10 23 DNA Artificial Sequence Synthetic10 ctcgaggcgg caaacccgtt gcg 23 11 1451 DNA Homo sapiens 11 aagctttggagctaagccag caatggtaga gggaagattc tgcacgtccc ttccaggcgg 60 cctccccgtcaccacccccc ccaacccgcc ccgaccggag ctgagagtaa ttcatacaaa 120 aggactcgcccctgccttgg ggaatcccag ggaccgtcgt taaactccca ctaacgtaga 180 acccagagatcgctgcgttc ccgccccctc acccgcccgc tctcgtcatc actgaggtgg 240 agaagagccatgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 300 tccccgagaagttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg 360 gggtaaactgggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 420 aacccgtatataagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc 480 agaacacaggtaagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc 540 ccttgcgtgccttgaattac ttccacgccc ctggctgcag tacgtgattc ttgatcccga 600 gcttcgggttggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc 660 tcgtgcttgagttgaggcct ggcctgggcg ctggggcccc cgcgtgcgaa tctggtggca 720 ccttcgcgcctgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc 780 tgctgcgacgctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac 840 tggtatttcggtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg 900 ttcggcgaggcggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc 960 tggccggcctgctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc 1020 aaggctggcccggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc 1080 tgcagggagctcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac 1140 acaaaggaaaagggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg 1200 ggcgccgtccaggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg 1260 gggggaggggttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag 1320 gccagcttggcacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg 1380 gttcattctcaagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg 1440 aaaactctag a1451 12 23 DNA Artificial Sequence Synthetic 12 tctagagttt tcacgacacctga 23 13 1289 DNA Mus musculus CDS (88)..(741) 13 ttacctcact gctttccggagcggtagcac ctcctccgcc ggcttcctcc tcagaccgct 60 ttttgccgcg agccgaccggtcccgtc atg ccg acc cgc agt ccc agc gtc gtg 114 Met Pro Thr Arg Ser ProSer Val Val 1 5 att agc gat gat gaa cca ggt tat gac cta gat ttg ttt tgtata cct 162 Ile Ser Asp Asp Glu Pro Gly Tyr Asp Leu Asp Leu Phe Cys IlePro 10 15 20 25 aat cat tat gcc gag gat ttg gaa aaa gtg ttt att cct catgga ctg 210 Asn His Tyr Ala Glu Asp Leu Glu Lys Val Phe Ile Pro His GlyLeu 30 35 40 att atg gac agg act gaa aga ctt gct cga gat gtc atg aag gagatg 258 Ile Met Asp Arg Thr Glu Arg Leu Ala Arg Asp Val Met Lys Glu Met45 50 55 gga ggc cat cac att gtg gcc ctc tgt gtg ctc aag ggg ggc tat aag306 Gly Gly His His Ile Val Ala Leu Cys Val Leu Lys Gly Gly Tyr Lys 6065 70 ttc ttt gct gac ctg ctg gat tac att aaa gca ctg aat aga aat agt354 Phe Phe Ala Asp Leu Leu Asp Tyr Ile Lys Ala Leu Asn Arg Asn Ser 7580 85 gat aga tcc att cct atg act gta gat ttt atc aga ctg aag agc tac402 Asp Arg Ser Ile Pro Met Thr Val Asp Phe Ile Arg Leu Lys Ser Tyr 9095 100 105 tgt aat gat cag tca acg ggg gac ata aaa gtt att ggt gga gatgat 450 Cys Asn Asp Gln Ser Thr Gly Asp Ile Lys Val Ile Gly Gly Asp Asp110 115 120 ctc tca act tta act gga aag aat gtc ttg att gtt gaa gat ataatt 498 Leu Ser Thr Leu Thr Gly Lys Asn Val Leu Ile Val Glu Asp Ile Ile125 130 135 gac act ggt aaa aca atg caa act ttg ctt tcc ctg gtt aag cagtac 546 Asp Thr Gly Lys Thr Met Gln Thr Leu Leu Ser Leu Val Lys Gln Tyr140 145 150 agc ccc aaa atg gtt aag gtt gca agc ttg ctg gtg aaa agg acctct 594 Ser Pro Lys Met Val Lys Val Ala Ser Leu Leu Val Lys Arg Thr Ser155 160 165 cga agt gtt gga tac agg cca gac ttt gtt gga ttt gaa att ccagac 642 Arg Ser Val Gly Tyr Arg Pro Asp Phe Val Gly Phe Glu Ile Pro Asp170 175 180 185 aag ttt gtt gtt gga tat gcc ctt gac tat aat gag tac ttcagg aat 690 Lys Phe Val Val Gly Tyr Ala Leu Asp Tyr Asn Glu Tyr Phe ArgAsn 190 195 200 ttg aat cac gtt tgt gtc att agt gaa act gga aaa gcc aaatac aaa 738 Leu Asn His Val Cys Val Ile Ser Glu Thr Gly Lys Ala Lys TyrLys 205 210 215 gcc taagatgagc gcaagttgaa tctgcaaata cgaggagtcctgttgatgtt 791 Ala gccagtaaaa ttagcaggtg ttctagtcct gtggccatctgcctagtaaa gctttttgca 851 tgaaccttct atgaatgtta ctgttttatt tttagaaatgtcagttgctg cgtccccaga 911 cttttgattt gcactatgag cctataggcc agcctaccctctggtagatt gtcgcttatc 971 ttgtaagaaa aacaaatctc ttaaattacc acttttaaataataatactg agattgtatc 1031 tgtaagaagg atttaaagag aagctatatt agttttttaattggtatttt aatttttata 1091 tattcaggag agaaagatgt gattgatatt gttaatttagacgagtctga agctctcgat 1151 ttcctatcag taacagcatc taagaggttt tgctcagtggaataaacatg tttcagcagt 1211 gttggctgta ttttcccact ttcagtaaat cgttgtcaacagttcctttt aaatgcaaat 1271 aaataaattc taaaaatt 1289 14 218 PRT Musmusculus 14 Met Pro Thr Arg Ser Pro Ser Val Val Ile Ser Asp Asp Glu ProGly 1 5 10 15 Tyr Asp Leu Asp Leu Phe Cys Ile Pro Asn His Tyr Ala GluAsp Leu 20 25 30 Glu Lys Val Phe Ile Pro His Gly Leu Ile Met Asp Arg ThrGlu Arg 35 40 45 Leu Ala Arg Asp Val Met Lys Glu Met Gly Gly His His IleVal Ala 50 55 60 Leu Cys Val Leu Lys Gly Gly Tyr Lys Phe Phe Ala Asp LeuLeu Asp 65 70 75 80 Tyr Ile Lys Ala Leu Asn Arg Asn Ser Asp Arg Ser IlePro Met Thr 85 90 95 Val Asp Phe Ile Arg Leu Lys Ser Tyr Cys Asn Asp GlnSer Thr Gly 100 105 110 Asp Ile Lys Val Ile Gly Gly Asp Asp Leu Ser ThrLeu Thr Gly Lys 115 120 125 Asn Val Leu Ile Val Glu Asp Ile Ile Asp ThrGly Lys Thr Met Gln 130 135 140 Thr Leu Leu Ser Leu Val Lys Gln Tyr SerPro Lys Met Val Lys Val 145 150 155 160 Ala Ser Leu Leu Val Lys Arg ThrSer Arg Ser Val Gly Tyr Arg Pro 165 170 175 Asp Phe Val Gly Phe Glu IlePro Asp Lys Phe Val Val Gly Tyr Ala 180 185 190 Leu Asp Tyr Asn Glu TyrPhe Arg Asn Leu Asn His Val Cys Val Ile 195 200 205 Ser Glu Thr Gly LysAla Lys Tyr Lys Ala 210 215 15 40 DNA Artificial Sequence Synthetic 15gcatgcgcgc ggccgcggag gctttttttt tttttttttt 40 16 27 DNA ArtificialSequence Synthetic 16 cggcaacgcg tgccatcatg gttcgac 27 17 30 DNAArtificial Sequence Synthetic 17 cggcagcggc cgcatagatc taaagccagc 30 18671 DNA Mus musculus CDS (13)..(573) 18 acgcgtgcca tc atg gtt cga ccattg aac tgc atc gtc gcc gtg tcc caa 51 Met Val Arg Pro Leu Asn Cys IleVal Ala Val Ser Gln 1 5 10 aat atg ggg att ggc aag aac gga gac cta ccctgg cct ccg ctc agg 99 Asn Met Gly Ile Gly Lys Asn Gly Asp Leu Pro TrpPro Pro Leu Arg 15 20 25 aac gag ttc aag tac ttc caa aga atg acc aca acctct tca gtg gaa 147 Asn Glu Phe Lys Tyr Phe Gln Arg Met Thr Thr Thr SerSer Val Glu 30 35 40 45 ggt aaa cag aat ctg gtg att atg ggt agg aaa acctgg ttc tcc att 195 Gly Lys Gln Asn Leu Val Ile Met Gly Arg Lys Thr TrpPhe Ser Ile 50 55 60 cct gag aag aat cga cct tta aag gac aga att aat atagtt ctc agt 243 Pro Glu Lys Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile ValLeu Ser 65 70 75 aga gaa ctc aaa gaa cca cca cga gga gct cat ttt ctt gccaaa agt 291 Arg Glu Leu Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala LysSer 80 85 90 ttg gat gat gcc tta aga ctt att gaa caa ccg gaa ttg gca agtaaa 339 Leu Asp Asp Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys95 100 105 gta gac atg gtt tgg ata gtc gga ggc agt tct gtt tac cag gaagcc 387 Val Asp Met Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala110 115 120 125 atg aat caa cca ggc cac ctt aga ctc ttt gtg aca agg atcatg cag 435 Met Asn Gln Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile MetGln 130 135 140 gaa ttt gaa agt gac acg ttt ttc cca gaa att gat ttg gggaaa tat 483 Glu Phe Glu Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly LysTyr 145 150 155 aaa ctt ctc cca gaa tac cca ggc gtc ctc tct gag gtc caggag gaa 531 Lys Leu Leu Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln GluGlu 160 165 170 aaa ggc atc aag tat aag ttt gaa gtc tac gag aag aaa gac573 Lys Gly Ile Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp 175 180 185taacaggaag atgctttcaa gttctctgct cccctcctaa agctatgcat ttttataaga 633ccatgggact tttgctggct ttagatctat gcggccgc 671 19 187 PRT Mus musculus 19Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 1015 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 2530 Lys Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 4045 Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 5560 Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu 65 7075 80 Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp 8590 95 Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp Met100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met AsnGln 115 120 125 Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln GluPhe Glu 130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys TyrLys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Glu Val GlnGlu Glu Lys Gly Ile 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys LysAsp 180 185 20 34 DNA Artificial Sequence Synthetic 20 atatatctagaccaccatgc ctggctcagc actg 34 21 35 DNA Artificial Sequence Synthetic 21attattgcgg ccgcttagct tttcattttg atcat 35 22 134 DNA Artificial SequenceSynthetic 22 ggtctagagc caaataaagg aagtggaacc acttcaggta ctacccgtcttctatctggg 60 cacacgtgtt tcacgttgac aggtttgctt gggacgctag taaccatgggcttgctgact 120 taggcatcga attc 134 23 134 DNA Artificial SequenceSynthetic 23 gaattcgatg cctaagtcag caagcccatg gttactagcg tcccaagcaaacctgtcaac 60 gtgaaacacg tgtgcccaga tagaagacgg gtagtacctg aagtggttccacttccttta 120 tttggctcta gacc 134 24 300 DNA Artificial SequenceSynthetic 24 taatacgact cactataggg cgaattggag ctccaccgcg gtggcggccgctctagaact 60 agtggatccc ccgggctgca ggaattcgat ggtctagagc caaataaaggaagtggaacc 120 acttcaggta ctacccgtct tctatctggg cacacgtgtt tcacgttgacaggtttgctt 180 gggacgctag taaccatggg cttgctgact taggcatcga attcatcaagcttatcgata 240 ccgtcgacct cgaggggggg cccggtaccc agcttttgtt ccctttagtgagggttaatt 300 25 28 DNA Artificial Sequence Synthetic 25 ccacttcctttatttgggag agggcttg 28 26 747 DNA Artificial Sequence Synthetic 26 atggcc ata agt gga gtc cct gtg cta gga ttt ttc atc ata gct gtg 48 Met AlaIle Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 ctgatg agc gct cag gaa tca tgg gct atc aaa gaa gaa cat gtg atc 96 Leu MetSer Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile 20 25 30 atc caggcc gag ttc tat ctg aat cct gac caa tca ggc gag ttt atg 144 Ile Gln AlaGlu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 ttt gac tttgat ggt gat gag att ttc cat gtg gat atg gca aag aag 192 Phe Asp Phe AspGly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys 50 55 60 gag acg gtc tggcgg ctt gaa gaa ttt gga cga ttt gcc agc ttt gag 240 Glu Thr Val Trp ArgLeu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 gct caa ggt gcattg gcc aac ata gct gtg gac aaa gcc aac ttg gaa 288 Ala Gln Gly Ala LeuAla Asn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 atc atg aca aag cgctcc aac tat act ccg atc acc aat gta cct cca 336 Ile Met Thr Lys Arg SerAsn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 gag gta act gtg ctcacg aac agc cct gtg gaa ctg aga gag ccc aac 384 Glu Val Thr Val Leu ThrAsn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 gtc ctc atc tgt ttcata gac aag ttc acc cca cca gtg gtc aat gtc 432 Val Leu Ile Cys Phe IleAsp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 acg tgg ctt cga aatgga aaa cct gtc acc aca gga gtg tca gag aca 480 Thr Trp Leu Arg Asn GlyLys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 gtc ttc ctg cccagg gaa gac cac ctt ttc cgc aag ttc cac tat ctc 528 Val Phe Leu Pro ArgGlu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 ccc ttc ctg ccctca act gag gac gtt tac gac tgc agg gtg gag cac 576 Pro Phe Leu Pro SerThr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190 tgg ggc ttg gatgag cct ctt ctc aag cac tgg gag ttt gat gct cca 624 Trp Gly Leu Asp GluPro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200 205 agc cct ctc ccaaat aaa gga agt gga acc act tca ggt act acc cgt 672 Ser Pro Leu Pro AsnLys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg 210 215 220 ctt cta tct gggcac acg tgt ttc acg ttg aca ggt ttg ctt ggg acg 720 Leu Leu Ser Gly HisThr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr 225 230 235 240 cta gta accatg ggc ttg ctg act tag 747 Leu Val Thr Met Gly Leu Leu Thr 245 27 248PRT Artificial Sequence Synthetic 27 Met Ala Ile Ser Gly Val Pro Val LeuGly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser TrpAla Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr Leu AsnPro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp Glu IlePhe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu Glu GluPhe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 Ala Gln Gly Ala Leu Ala AsnIle Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg Ser AsnTyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val Leu ThrAsn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile Cys PheIle Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp Leu ArgAsn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 Val PheLeu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 ProPhe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200205 Ser Pro Leu Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg 210215 220 Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr225 230 235 240 Leu Val Thr Met Gly Leu Leu Thr 245 28 28 DNA ArtificialSequence Synthetic 28 ccacttcctt tatttggtgc agattcag 28 29 786 DNAArtificial Sequence Synthetic 29 atg gtg tgt ctg aag ctc cct gga ggc tcctgc atg aca gcg ctg aca 48 Met Val Cys Leu Lys Leu Pro Gly Gly Ser CysMet Thr Ala Leu Thr 1 5 10 15 gtg aca ctg atg gtg ctg agc tcc cga ctggct ttg gct ggg gac acc 96 Val Thr Leu Met Val Leu Ser Ser Arg Leu AlaLeu Ala Gly Asp Thr 20 25 30 cga cca cgt ttc ttg tgg cag ctt aag ttt gaatgt cat ttc ttc aat 144 Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Glu CysHis Phe Phe Asn 35 40 45 ggg acg gag cgg gtg cgg ttg ctg gaa aga tgc atctat aac caa gag 192 Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile TyrAsn Gln Glu 50 55 60 gag tcc gtg cgc ttc gac agc gac gtg ggg gag tac cgggcg gtt gag 240 Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg AlaVal Glu 65 70 75 80 gag ctg ggg cgg cct gat gcc gag tac tgg aac agc cagaag gac ctc 288 Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln LysAsp Leu 85 90 95 ctg gag cag aag cgg ggc cag gtg gac aat tac tgc aga cacaac tac 336 Leu Glu Gln Lys Arg Gly Gln Val Asp Asn Tyr Cys Arg His AsnTyr 100 105 110 ggg gtt ggt gag agc ttc aca gtg cag cgg cga gtt gag cctaag gtg 384 Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val Glu Pro LysVal 115 120 125 act gtg tat cct tca aag acc cag ccc ctg cag cac cac aacctc ctg 432 Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gln His His Asn LeuLeu 130 135 140 gtc tgc tct gtg agt ggt ttc tat cca ggc agc att gaa gtcagg tgg 480 Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val ArgTrp 145 150 155 160 ttc cgg aac ggc cag gaa gag aag gct ggg gtg gtg tccacg ggc ctg 528 Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser ThrGly Leu 165 170 175 atc cag aat gga gat tgg acc ttc cag acc ctg gtg atgctg gaa ata 576 Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met LeuGlu Ile 180 185 190 gtt cct cgg agt gga gag gtt tac acc tgc caa gtg gagcac cca agt 624 Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu HisPro Ser 195 200 205 gtg acg agc cct ctc aca gtg gaa tgg aga gca cgg tctgaa tct gca 672 Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser GluSer Ala 210 215 220 cca aat aaa gga agt gga acc act tca ggt act acc cgtctt cta tct 720 Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg LeuLeu Ser 225 230 235 240 ggg cac acg tgt ttc acg ttg aca ggt ttg ctt gggacg cta gta acc 768 Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly ThrLeu Val Thr 245 250 255 atg ggc ttg ctg act tag 786 Met Gly Leu Leu Thr260 30 261 PRT Artificial Sequence Synthetic 30 Met Val Cys Leu Lys LeuPro Gly Gly Ser Cys Met Thr Ala Leu Thr 1 5 10 15 Val Thr Leu Met ValLeu Ser Ser Arg Leu Ala Leu Ala Gly Asp Thr 20 25 30 Arg Pro Arg Phe LeuTrp Gln Leu Lys Phe Glu Cys His Phe Phe Asn 35 40 45 Gly Thr Glu Arg ValArg Leu Leu Glu Arg Cys Ile Tyr Asn Gln Glu 50 55 60 Glu Ser Val Arg PheAsp Ser Asp Val Gly Glu Tyr Arg Ala Val Glu 65 70 75 80 Glu Leu Gly ArgPro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu 85 90 95 Leu Glu Gln LysArg Gly Gln Val Asp Asn Tyr Cys Arg His Asn Tyr 100 105 110 Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg Val Glu Pro Lys Val 115 120 125 Thr ValTyr Pro Ser Lys Thr Gln Pro Leu Gln His His Asn Leu Leu 130 135 140 ValCys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp 145 150 155160 Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu 165170 175 Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Ile180 185 190 Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His ProSer 195 200 205 Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser GluSer Ala 210 215 220 Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr ArgLeu Leu Ser 225 230 235 240 Gly His Thr Cys Phe Thr Leu Thr Gly Leu LeuGly Thr Leu Val Thr 245 250 255 Met Gly Leu Leu Thr 260 31 189 DNAArtificial Sequence Synthetic 31 ttg gat cca cga tcg ttt cta ttg cgc aatcca aat gat aag tac gaa 48 Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn ProAsn Asp Lys Tyr Glu 1 5 10 15 cca ttt tgg gaa gat act aca gag aac gtggtg tgt gcc ctg ggc ctg 96 Pro Phe Trp Glu Asp Thr Thr Glu Asn Val ValCys Ala Leu Gly Leu 20 25 30 act gtg ggt ctg gtg ggc atc att att ggg accatc ttc atc atc aag 144 Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr IlePhe Ile Ile Lys 35 40 45 gga gtg cgc aaa agc aat gca gca gaa cgc agg gggcct ctg taa 189 Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu50 55 60 32 62 PRT Artificial Sequence Synthetic 32 Leu Asp Pro Arg SerPhe Leu Leu Arg Asn Pro Asn Asp Lys Tyr Glu 1 5 10 15 Pro Phe Trp GluAsp Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu 20 25 30 Thr Val Gly LeuVal Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys 35 40 45 Gly Val Arg LysSer Asn Ala Ala Glu Arg Arg Gly Pro Leu 50 55 60 33 192 DNA ArtificialSequence Synthetic 33 ttg gat cca cga tcg ttt cta ttg cgc aat cca aatgat aag tac gaa 48 Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pro Asn AspLys Tyr Glu 1 5 10 15 cca ttt tgg gaa gat cag agc aag atg ctg agt ggagtc ggg ggc ttc 96 Pro Phe Trp Glu Asp Gln Ser Lys Met Leu Ser Gly ValGly Gly Phe 20 25 30 gtg ctg ggc ctg ctc ttc ctt ggg gcc ggg ctg ttc atctac ttc agg 144 Val Leu Gly Leu Leu Phe Leu Gly Ala Gly Leu Phe Ile TyrPhe Arg 35 40 45 aat cag aaa gga cac tct gga ctt cag cca aca gga ttc ctgagc tga 192 Asn Gln Lys Gly His Ser Gly Leu Gln Pro Thr Gly Phe Leu Ser50 55 60 34 63 PRT Artificial Sequence Synthetic 34 Leu Asp Pro Arg SerPhe Leu Leu Arg Asn Pro Asn Asp Lys Tyr Glu 1 5 10 15 Pro Phe Trp GluAsp Gln Ser Lys Met Leu Ser Gly Val Gly Gly Phe 20 25 30 Val Leu Gly LeuLeu Phe Leu Gly Ala Gly Leu Phe Ile Tyr Phe Arg 35 40 45 Asn Gln Lys GlyHis Ser Gly Leu Gln Pro Thr Gly Phe Leu Ser 50 55 60 35 39 DNAArtificial Sequence Synthetic 35 cgatcgtgga tccaagttta ggttcgtatctgtttcaaa 39 36 34 DNA Artificial Sequence Synthetic 36 cgatcgaggatccaagatgg tggcagacag gacc 34 37 32 DNA Artificial Sequence Synthetic 37acgcgtccac catggccata agtggagtcc ct 32 38 28 DNA Artificial SequenceSynthetic 38 ggatccaact ctgtagtctc tgggagag 28 39 32 DNA ArtificialSequence Synthetic 39 acgcgtccac catggtgtgt ctgaagctcc tg 32 40 29 DNAArtificial Sequence Synthetic 40 ggatccaact tgctctgtgc agattcaga 29 41292 DNA Homo sapiens 41 gaattctttt ttgcgtgtgg cagttttaag ttattagtttttaaaatcag tactttttaa 60 tggaaacaac ttgaccaaaa atttgtcaca gaattttgagacccattaaa aaagttaaat 120 gagaaacctg tgtgttcctt tggtcaacac cgagacatttaggtgaaaga catctaattc 180 tggttttacg aatctggaaa cttcttgaaa atgtaattcttgagttaaca cttctgggtg 240 gagaataggg ttgttttccc cccacataat tggaaggggaaggaatatcg at 292 42 20 DNA Artificial Sequence Synthetic 42 tcgatggcgcgccttaatta 20 43 20 DNA Artificial Sequence Synthetic 43 agcttaattaaggcgcgcca 20 44 1147 DNA Artificial Sequence Synthetic 44 gcggccgcgtcgaccaaggg ccccagcgtg ttccccctgg ccccctgctc ccgcagcacc 60 agcggcggcaccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 120 gtgagctggaacagcggcgc cctgaccagc ggcgtccaca ccttccccgc cgtgctgcag 180 tccagcggcctgtactccct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc 240 cagacctacacctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caagcgcgtg 300 gagctgaagacccccctggg cgacaccacc cacacctgcc cccgctgccc cgagcccaag 360 agctgcgacacccctccccc ctgcccccgc tgccccgagc ccaagagctg cgacacccct 420 cccccctgcccccgctgccc cgagcccaag agctgcgaca cccctccccc ctgcccccgc 480 tgccccgcccccgagctgct gggcggcccc agcgtgttcc tgttcccccc caagcccaag 540 gacaccctgatgatctcccg cacccccgag gtgacctgcg tggtggtgga cgtgagccac 600 gaggaccccgaggtgcagtt caagtggtac gtggacggcg tggaggtgca taacgccaag 660 accaagccccgcgaggagca gtacaacagc accttccgcg tggtgagcgt gctgaccgtg 720 ctgcaccaggactggctgaa cggcaaggag tacaagtgca aggtgagcaa caaggccctg 780 cccgcccccatcgagaagac catctccaag accaagggcc agccccgcga gccccaggtg 840 tacaccctgccccccagccg cgaggagatg accaagaacc aggtgagcct gacctgcctg 900 gtgaagggcttctaccccag cgacatcgcc gtggagtggg agagcagcgg ccagcccgag 960 aacaactacaacaccacccc ccccatgctg gacagcgacg gcagcttctt cctgtacagc 1020 aagctgaccgtggacaagag ccgctggcag cagggcaaca tcttctcctg cagcgtgatg 1080 catgaggccctgcacaaccg cttcacccag aagagcctga gcctgagccc cggcaagtga 1140 tagatct 114745 377 PRT Homo sapiens 45 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu GlnSer Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser SerSer Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys ProSer Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu GlyAsp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser CysAsp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser CysAsp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser CysAsp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu LeuLeu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys AspThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val ValAsp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 ValAsp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys GlyGln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg GluGlu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly GlnPro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp SerAsp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys SerArg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His GluAla Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser ProGly Lys 370 375 46 999 DNA Artificial Sequence Synthetic 46 gcggccgcgcgtcgaccaag ggccccagcg tgttccccct ggccccctgc agccgcagca 60 ccagcgagagcaccgccgcc ctgggctgcc tggtgaagga ctacttcccc gagcccgtga 120 ccgtgagctggaacagcggc gccctgacca gcggcgtgca caccttcccc gccgtgctgc 180 agagcagcggcctgtactcc ctgagcagcg tggtgaccgt gcccagcagc agcctgggca 240 ccaagacctacacctgcaac gtggaccaca agcccagcaa caccaaggtg gacaagcgcg 300 tggagagcaagtacggcccc ccctgcccca gctgccccgc ccccgagttc ctgggcggcc 360 ccagcgtgttcctgttcccc cccaagccca aggacaccct gatgatcagc cgcacccccg 420 aggtgacctgcgtggtggtg gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt 480 acgtggacggcgtggaggtg cataacgcca agaccaagcc ccgcgaggag cagttcaaca 540 gcacctaccgcgtggtgagc gtgctgaccg tgctgcacca ggactggctg aacggcaagg 600 agtacaagtgcaaggtgtcc aacaagggcc tgcccagcag catcgagaag accatcagca 660 aggccaagggccagccccgc gagccccagg tgtacaccct gccccccagc caggaggaga 720 tgaccaagaaccaggtgagc ctgacctgcc tggtgaaggg cttctacccc agcgacatcg 780 ccgtggagtgggagagcaac ggccagcccg agaacaacta caagaccacc ccccccgtgc 840 tggacagcgacggcagcttc ttcctgtaca gccgcctgac cgtggacaag agccgctggc 900 aggagggcaacgtgttctcc tgctccgtga tgcatgaggc cctgcacaac cactacaccc 960 agaagagcctgagcctgagc ctgggcaagt gatagatct 999 47 327 PRT Homo sapiens 47 Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 SerThr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 PhePro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 GlyVal His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 LeuSer Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr ValAsp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro ProSer Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp GluSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr ValAsp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser ValMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 LeuSer Leu Ser Leu Gly Lys 325 48 337 DNA Artificial Sequence Synthetic 48gcggccgcac tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagctta 60agtccggaac cgccagcgtg gtgtgcctgc tgaacaactt ctacccccgc gaggccaagg 120tgcagtggaa ggtggacaac gccctccaga gcggcaactc ccaggagagc gtgaccgagc 180aggacagcaa ggacagcacc tacagcctga gcagcaccct gaccctgagc aaggccgact 240acgagaagca caaggtgtac gcctgcgagg tgacccatca gggcctgagc agccccgtga 300ccaagagctt caaccggggc gagtgctagt gagatct 337 49 106 PRT Homo sapiens 49Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 1015 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 2530 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 4045 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 5560 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 7075 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 8590 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 50 346 DNAArtificial Sequence Synthetic 50 gcggccgcac cgtcctaggt cagcccaaggcggcgcccag cgtgaccctg ttccccccca 60 gcagcgagga gctgcaggcc aacaaggccaccctggtgtg cctgatcagc gacttctacc 120 ccggggccgt gaccgtggcc tggaaggccgacagcagccc cgtgaaggcc ggcgtggaga 180 ccaccacccc cagcaagcag agcaacaacaagtacgccgc cagcagctac ctgagcctga 240 cccccgagca gtggaagagc caccgcagctacagctgcca ggtcacccac gagggcagca 300 ccgtggagaa gaccgtggcc cccaccgagtgcagctagtg agatct 346 51 109 PRT Homo sapiens 51 Thr Val Leu Gly Gln ProLys Ala Ala Pro Ser Val Thr Leu Phe Pro 1 5 10 15 Pro Ser Ser Glu GluLeu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu 20 25 30 Ile Ser Asp Phe TyrPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp 35 40 45 Ser Ser Pro Val LysAla Gly Val Glu Thr Thr Thr Pro Ser Lys Gln 50 55 60 Ser Asn Asn Lys TyrAla Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu 65 70 75 80 Gln Trp Lys SerHis Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly 85 90 95 Ser Thr Val GluLys Thr Val Ala Pro Thr Glu Cys Ser 100 105 52 38 DNA ArtificialSequence Synthetic 52 tctagaattc acgcgtccac catggactgg acctggag 38 53 41DNA Artificial Sequence Synthetic 53 tctagaattc acgcgtccac catggacacactttgctaca c 41 54 42 DNA Artificial Sequence Synthetic 54 tctagaattcacgcgtccac catggagttt gggctgagct gg 42 55 44 DNA Artificial SequenceSynthetic 55 tctagaattc acgcgtccac catgaaacac ctgtggttct tcct 44 56 41DNA Artificial Sequence Synthetic 56 tctagaattc acgcgtccac catggggtcaaccgccatcc t 41 57 44 DNA Artificial Sequence Synthetic 57 tctagaattcacgcgtccac catgtctgtc tccttcctca tctt 44 58 24 DNA Artificial SequenceSynthetic 58 gcctgagttc cacgacaccg tcac 24 59 24 DNA Artificial SequenceSynthetic 59 ggggaaaagg gttggggcgg atgc 24 60 39 DNA Artificial SequenceSynthetic 60 gaggggccct tggtcgacgc tgaggagacg gtgaccagg 39 61 40 DNAArtificial Sequence Synthetic 61 gaggggccct tggtcgacgc tgaagagacggtgaccattg 40 62 39 DNA Artificial Sequence Synthetic 62 gaggggcccttggtcgacgc tgaggagacg gtgaccgtg 39 63 45 DNA Artificial SequenceSynthetic 63 tctagaattc acgcgtccac catggacatg agggtccccg ctcag 45 64 40DNA Artificial Sequence Synthetic 64 tctagaattc acgcgtccac catgaggctccctgctcagc 40 65 42 DNA Artificial Sequence Synthetic 65 tctagaattcacgcgtccac catggaagcc ccagcgcagc tt 42 66 41 DNA Artificial SequenceSynthetic 66 tctagaattc acgcgtccac catggtgttg cagacccagg t 41 67 41 DNAArtificial Sequence Synthetic 67 tctagaattc acgcgtccac catggggtcccaggttcacc t 41 68 43 DNA Artificial Sequence Synthetic 68 tctagaattcacgcgtccac catgttgcca tcacaactca ttg 43 69 41 DNA Artificial SequenceSynthetic 69 tctagaattc acgcgtccac catggtgtcc ccgttgcaat t 41 70 34 DNAArtificial Sequence Synthetic 70 ggttccggac ttaagctgct catcagatgg cggg34 71 44 DNA Artificial Sequence Synthetic 71 tctagaattc acgcgtccaccatggcctgc tctcctctcc tcct 44 72 44 DNA Artificial Sequence Synthetic 72tctagaattc acgcgtccac catggcctgg gctctgctgc tcct 44 73 45 DNA ArtificialSequence Synthetic 73 tctagaattc acgcgtccac catggcctgg atccttctcc tcctc45 74 45 DNA Artificial Sequence Synthetic 74 tctagaattc acgcgtccaccatggcctgg acccctctct ggctc 45 75 41 DNA Artificial Sequence Synthetic75 tctagaattc acgcgtccac catggcctgg gccccactac t 41 76 44 DNA ArtificialSequence Synthetic 76 tctagaattc acgcgtccac catggcctgg atgatgcttc tcct44 77 29 DNA Artificial Sequence Synthetic 77 ggcgccgcct tgggctgacctaggacggt 29 78 23 DNA Artificial Sequence Synthetic 78 gaattcttttttgcgtgtgg cag 23 79 21 DNA Artificial Sequence Synthetic 79 atcgatattccttccccttc c 21 80 17 DNA Homo sapiens misc_feature (17)..(17) N at thisposition can be a, c, t, or g, and can be repeated 18 to 21 times. 80tctagaattc acgcgtn 17

1. A multivalent vaccine comprising at least two recombinant variableregions of immunoglobulin molecules derived from B-cell lymphoma cells,wherein said cells express at least two different immunoglobulinmolecules, said immunoglobulin molecules differing by at least oneidiotope.
 2. The vaccine of claim 1, wherein said vaccine comprises atleast two recombinant immunoglobulin molecules comprising saidrecombinant variable regions derived from said lymphoma cells.
 3. Thevaccine of claim 2, wherein said recombinant immunoglobulin moleculesare covalently linked to an immune-enhancing cytokine.
 4. The vaccine ofclaim 3, wherein said cytokine is selected from the group consisting ofgranulocyte-macrophage colony stimulating factor, interleukin-2 andinterleukin-4.
 5. The multivalent vaccine of claim 1 further comprisingat least one pharmaceutically acceptable excipient.
 6. The multivalentvaccine of claim 1 further comprising an adjuvant.
 7. A method ofproducing a vaccine for treatment of B-cell lymphoma comprising: a)providing: i) malignant cells isolated from a patient having a B-celllymphoma; ii) an amplification vector comprising a recombinantoligonucleotide having a sequence encoding a first inhibitable enzymeoperably linked to a heterologous promoter; iii) a eukaryotic parentcell line; b) isolating from said malignant cells nucleotide sequencesencoding at least one V_(H) region and at least one V_(L) region, saidV_(H) and V_(L) regions derived from immunoglobulin molecules expressedby said malignant cells; c) inserting said nucleotide sequences encodingsaid V_(H) and V_(L) regions into at least one expression vector; d)introducing said at least one expression vector and said amplificationvector into said parent cell to generate one or more transformed cells;e) growing said transformed cell in a first aqueous solution containingan inhibitor capable of inhibiting said inhibitable enzyme wherein theconcentration of said inhibitor present in said first aqueous solutionis sufficient to prevent growth of said parent cell line; and f)identifying a transformed cell capable of growth in said first aqueoussolution, wherein said transformed cell capable of growth expresses saidVH and VL regions.
 8. The methods of claim 7, wherein transformed cellcapable of growth contains an amplified number of copies of saidexpression vector and an amplified number of copies of saidamplification vector.
 9. The method of claim 7, wherein nucleotidesequences encoding said V_(H) and V_(L) regions comprise at least twoV_(H) and at least two V_(L) regions.
 10. The method of claim 7, whereinsaid parent cell line is a T lymphoid cell line.
 11. The method of claim7, wherein said parent cell line contains an endogenous gene encoding asecond inhibitable enzyme.
 12. The method of claim 11, wherein saidsecond inhibitable enzyme is selected from the group consisting ofdihydrofolate reductase, glutamine synthetase, adenosine deaminase,asparagine synthetase.
 13. The method of claim 7, wherein saidconcentration of inhibitor present in said first aqueous solution isfour to six-fold the concentration required to prevent the growth ofsaid parent cell line.
 14. The method of claim 7, wherein said first andsaid second inhibitable enzyme are the same.
 15. The method of claim 7,further comprising providing a selection vector encoding a selectablegene product which is introduced into said parent cell line togetherwith said expression vector and said amplification vector.
 16. Themethod of claim 15, wherein said selection vector encodes an activehypoxanthine guanine phosphoribosyltransferase.
 17. The method of claim16, wherein said aqueous solution which requires the expression of saidselectable gene product comprises hypoxanthine and azaserine.
 18. Themethod of claim 15, further comprising following the introduction ofsaid vectors the additional step of growing said transformed cell in asecond aqueous solution which requires the expression of said selectablegene product prior to growing said transformed cell said first aqueoussolution containing an inhibitor capable of inhibiting said inhibitableenzyme.
 19. The method of claim 7, wherein said amplification vectorencodes an active enzyme selected from the group consisting ofdihydrofolate reductase, glutamine synthetase, adenosine deaminase,asparagine synthetase.
 20. The method of claim 19, wherein saidinhibitor is selected from the group consisting of methotrexate,2′-deoxycoformycin, methionine sulphoximine, albizziin and β-aspartylhydroxamate.
 21. A method of treating B-cell lymphoma, comprising: a)providing: i) a subject having a B-cell lymphoma; ii) a multivalentvaccine comprising at least two recombinant variable regions ofimmunoglobulin molecules derived from said subjects's B-cell lymphomacells, wherein said cells express at least two different immunoglobulinmolecules, said immunoglobulin molecules differing by at least oneidiotope; b) administering said multivalent vaccine to said subject. 22.The method of claim 21, wherein said vaccine comprises at least tworecombinant immunoglobulin molecules comprising said recombinantvariable regions derived from said lymphoma cells.
 23. The method ofclaim 21, wherein said vaccine further comprises an adjuvant.
 24. Themethod of claim 22, wherein said adjuvant is Syntex adjuvant formulation1.